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Molecular Structure and Functionof the Glycine Receptor Chloride Channel

JOSEPH W. LYNCH

School of Biomedical Sciences, University of Queensland, Brisbane, Australia

I. Introduction 1052 A. Scope of this review 1052B. Glycine as an inhibitory (and excitatory) neurotransmitter 1052

II. Diversity, Distribution, and Function of Glycine Receptor Subunits 1052 A. Molecular diversity 1052B. Distribution and function in the rat nervous system 1053

C. Distribution and function in other tissues 1055III. Structure and Assembly 1056 A. General structural features 1056B. Transmembrane domains 1056C. NH2-terminal ligand-binding domain 1059D. Large intracellular domain 1062E. Receptor assembly 1063

IV. Structure and Function of the Pore 1064 A. Functional properties of the pore 1064B. Molecular determinants of ion selectivity and conductance 1064C. The channel activation gate 1066D. Molecular mechanisms of desensitization 1066

V. Agonist Binding and Receptor Activation 1066 A. Introduction 1066B. Kinetic models of glycine receptor gating 1067

C. Agonist binding 1068D. Structural changes accompanying activation 1070 VI. Glycine Receptor Modulation 1072

A. Phosphorylation 1072B. Modulators of possible physiological relevance 1073C. Molecular pharmacology 1076

VII. Glycine Receptor Channelopathies 1082 A. Human startle disease 1082B. Murine startle syndromes 1083C. Bovine myoclonus 1084

VIII. Outlook 1084

Lynch, Joseph W. Molecular Structure and Function of the Glycine Receptor Chloride Channel. Physiol Rev 84:

1051–1095, 2004; 10.1152/physrev.00042.2003.—The glycine receptor chloride channel (GlyR) is a member of thenicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise fivesubunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitoryneurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other

physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four ␣-subunits (␣1 to␣4) and one ␤-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR

pharmacology. A developmental switch from ␣2 to ␣1␤ is completed by around postnatal day 20 in the rat. The␤-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin.The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information hasrecently emerged concerning GlyR molecular structure and function. Most of the information has been obtainedfrom homomeric ␣1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritablemutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent

Physiol Rev

84: 1051–1095, 2004; 10.1152/physrev.00042.2003.

www.prv.org 10510031-9333/04 $15.00 Copyright © 2004 the American Physiological Society



modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide

inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of

muscle relaxant and peripheral analgesic drugs.

I. INTRODUCTION

A. Scope of This Review

Ligand-gated ion channels permit cells to respond

rapidly to changes in their external environment. They are

particularly well known for mediating fast neurotransmis-

sion in the nervous system. The glycine receptor (GlyR) is

a membrane-embedded protein that contains an integral

ClϪ-selective pore. When glycine binds to its site on the

external receptor surface, the pore opens allowing ClϪ to

passively diffuse across the membrane. The GlyR is a

member of the pentameric ligand-gated ion channel

(LGIC) family, of which the nicotinic acetylcholine recep-tor cation channel (nAChR) is the prototypical member.

Other members of this family include the cation-perme-

able serotonin type 3 receptor (5-HT3R), anion-permeable

GABA type A and C receptors (GABA A R and GABA CR),

recently identified cation-permeable zinc and GABA re-

ceptors (34, 86), as well as invertebrate anion-permeable

glutamate and histidine receptors (130). Note that glycine

also directly activates a cation-selective ion channel of the

excitatory glutamate receptor family (62). The structural

and functional properties of this receptor class have re-

cently been reviewed (94) and are not considered here.

Glycine was first proposed as an inhibitory neuro-

transmitter on the basis of a detailed analysis of its dis-tribution in the spinal cord (16). Subsequent electrophys-

iological studies demonstrated a strychnine-sensitive hy-

perpolarizing action of glycine on spinal neurons (80,

395). This hyperpolarization was soon discovered to be

mediated by an increase in ClϪ conductance (81, 82, 396).

The receptors responsible for these actions were subse-

quently purified by strychnine af finity chromatography

(291, 292), and the first GlyR subunit was cloned in 1987

(138).

Current research into the GlyR can be divided into

two major strands. The first involves the investigation of

the molecular mechanisms of GlyR traf ficking and clus-tering at synapses. This area is currently the subject of

intense investigation, and recent progress has been cov-

ered in several authoritative reviews (189, 190, 219). The

second research strand is concerned with understanding

the molecular structure and function of the GlyR. Re-

search has intensified in this area over the past few years,

and the purpose of this review is to present a coherent

view of recent findings. Much of our understanding of

GlyR structure-function has been gained by comparison

with the structurally homologous nAChR. Hence, this re-

view makes frequent references to research on the

nAChR, particularly in areas where knowledge of the

GlyR is deficient.

B. Glycine as an Inhibitory

(and Excitatory) Neurotransmitter

When the GlyR is activated, the resulting ClϪ flux

moves the membrane potential rapidly toward the ClϪ

equilibrium potential. Depending on the value of the equi-

librium potential relative to the cell resting potential, the

ClϪ flux may cause either a depolarization or a hyperpo-

larization. The GlyR is generally known as an inhibitory

receptor because the ClϪ equilibrium potential is usually

close to or more negative than the cell resting potential.Subthreshold depolarizations can inhibit neuronal firing if

they are accompanied by an increase in membrane con-

ductance that shorts out excitatory responses, a phenom-

enon termed “shunting inhibition.” However, in embry-

onic neurons, the intracellular ClϪ concentration is raised

substantially, with the effect that GlyR activation causes a

strong, suprathreshold depolarization. These large gly-

cine-induced depolarizations gate a calcium influx that is

necessary for the development of numerous specializa-

tions, including glycinergic synapses (190). The switch to

the mature neuron phenotype is mediated by the expres-

sion of a K ϩ

-ClϪ

cotransporter, KCC2, which lowers theinternal ClϪ concentration, thereby shifting the ClϪ equi-

librium potential to more negative values and converting

the actions of the GlyR from excitatory to inhibitory

(355).

II. DIVERSITY, DISTRIBUTION, AND FUNCTION

OF GLYCINE RECEPTOR SUBUNITS

A. Molecular Diversity

Betz and colleagues (291, 292) originally purified therat spinal cord GlyR by af finity chromatography on amino-

strychnine-agarose columns. Oligonucleotides designed

from peptide sequences of purified receptors were then

used to probe a rat spinal cord cDNA library, resulting in

isolation of cDNA clones corresponding to the 48 kDa

(␣1) and 58 kDa (␤) subunits (137, 138). Subsequently,

cDNAs of the rat ␣2- and ␣3-subunits were cloned by

homology screening (9, 199, 201). The ␣4-subunit, which

does not appear to exist in the rat or human, was first

identified in the mouse (254) and has subsequently been

found in the chick (157) and zebra fish (91). While the

1052 JOSEPH W. LYNCH

Physiol Rev • VOL 84 • OCTOBER 2004 • www.prv.org



␣-subunit genes are highly homologous, with primary

structures displaying 80 –90% amino acid sequence iden-

tity, the ␤-subunit has a sequence similarity of ϳ47% with

the ␣1-subunit (137).

The rat ␣1-subunit has a splice variant, termed ␣1ins,

which contains an eight-amino acid insert in the large

intracellular loop (244) that contains a possible phosphorylation site (see sect. VI A). Alternative splicing of the rat

␣2-subunit generates two splice variants, ␣2A and ␣2B

(199, 201). The ␣2B-variant differs from ␣2A by the V58I

and T59A amino acid substitutions. Another version of the

␣2-subunit, termed ␣2*, incorporates a single amino acid

substitution (G167E) that confers strychnine insensitivity

(202). Two transcripts of the human ␣3-gene, termed ␣3L

and ␣3K, have been characterized (280). The ␣3K-variant

lacks a 15-amino acid segment in the large intracellular

loop that exists in both the rat ␣3- and the human ␣3L-

subunits. A splice variant of the zebra fish ␣4-subunit con-

tains a 15-amino acid insert in the ligand-binding domain(91). Although a human ␤-subunit gene polymorphism has

been described (264), it does not appear to result in a

coding mutation.

All GlyR genes cloned to date share a similar exon-

intron organization with the coding region spread over

nine exons (254, 264, 346). This common organization

suggests a phylogenetic gene duplication (345).

B. Distribution and Function in the Rat

Nervous System

1. Distribution of functional GlyRs A ) DISTRIBUTION OF STRYCHNINE AND GLYCINE BINDING SITES.

Autoradiographic localization of [3H]strychnine binding

sites was first studied by Zarbin et al. (431) in the rat.

Strychnine binding sites were shown to exist at high

levels in the spinal cord and medulla and at lower levels

in the pons, thalamus, and hypothalamus, while being

virtually absent in higher brain regions. In the spinal cord,

their distribution is relatively diffuse throughout the gray

matter. In contrast, GlyRs in the brain stem are highly

localized to discrete nuclei, notably the trigeminal nuclei,

the cuneate nucleus, the gracile nucleus, the hypoglossal

motor nucleus, the reticular nuclei, and cochlear nuclei(301). The retina, which also has a high concentration of

strychnine sites (297), is considered as a separate case,

below. Together, these areas comprise a subset of the

distribution as determined by glycine autoradiography

(270) or glycine immunoreactivity (310), a mismatch that

is understandable given that glycine is also associated

with glutamatergic synapses (270). An advantage of

strychnine autoradiography is that it reveals the presence

of surface-expressed receptors, but a disadvantage is that

its limited resolution does not permit the ultrastructural

localization of strychnine binding sites. Thus strychnine

autoradiography does not necessarily define the distribu-

tion of glycinergic synapses.

B) DISTRIBUTION OF GLYR IMMUNOREACTIVITY . Many studies

have examined the immunocytochemical localization of

GlyRs at the light and electron microscopic levels using

generic GlyR ␣-subunit monoclonal antibodies. At the

light microscopic level, there is a strong correlation be-tween the distribution patterns revealed by GlyR immu-

noreactivity and strychnine autoradiography (17, 370).

However, some significant differences have been ob-

served. First, immunolabeling reveals the existence of

GlyRs in the cerebellum (17, 361) and olfactory bulb

(383), whereas none was seen using strychnine binding

(431). Second, the substantia gelatinosa in the spinal cord

is strongly labeled by strychnine (431), but not by GlyR

antibodies (23). The reasons for the differential labeling

are yet to be clarified.

Electron microscopic immunoreactivity reveals that

GlyRs at central synapses are concentrated into regionsclosely apposed to presynaptic terminals (13, 23, 370, 371,

383), strongly suggesting a functional role. It is of interest

to note that this approach has demonstrated the colocal-

ization of GlyRs and GABA A Rs at individual postsynaptic

densities in the spinal cord (43, 127, 369) and cerebellum

(100).

C) DISTRIBUTION OF FUNCTIONAL GLYCINERGIC SYNAPSES. Most

central nervous system neurons are inhibited by glycine

(279). Of course, the mere presence of functional GlyRs,

especially on dissociated or cultured neurons, does not

imply a physiological role. However, it has recently been

proposed that the activation of nonsynaptic GlyRs in em-

bryonic cortical neurons may be important for develop-ment, and that taurine released from local glial cells may

be the endogenous ligand (114). Similarly, nonsynaptic

GlyRs on hippocampal CA3 neurons are proposed to be

held in a tonically active state by locally released taurine

or ␤-alanine (273). The idea that glycinergic ligands may

act on nonsynaptic GlyRs to mediate processes of physi-

ological importance certainly warrants further attention.

Traditionally, however, a functional role for GlyRs in

neurons has required the demonstration of strychnine-

sensitive synaptic currents.

There is abundant evidence for the existence of func-

tional glycinergic synapses in the retina (see below) inspinal cord motor reflex pathways (219) and in spinal

cord pain sensory pathways (4). Glycinergic neurotrans-

mission has also been demonstrated in various brain stem

nuclei. For example, it has been well characterized in

several brain stem nuclei of the central auditory path-

ways. In the medullary cochlear nucleus, which receive

inputs directly from the auditory nerve, glycinergic syn-

apses occur onto stellate cells (108) and bushy cells (230).

In the trapezoid body, a subsequent major relay station in

the auditory pathway, GlyRs are located presynaptically

at calyceal synapses onto principal cells (373). The prom-

THE GLYCINE RECEPTOR CHLORIDE CHANNEL 1053




inent output from this nuclei extends to the superior

olivary complex of the pons, where glycinergic synapses

are also found (194, 351). Functional glycinergic synapses

also exist on neurons in the medullary trigeminal (421),

abducens (325), and hypoglossal motor nuclei (248, 347,

375). In the cerebellum, glycinergic synapses mediate in-

hibitory neurotransmission between Lugaro cells andGolgi cells in the cerebellar cortex (93), and between

interneurons and principal cells in the deep cerebellar

nuclei (182). This list may expand as other brain stem

nuclei are characterized in detail.

It is relevant to note that glycine may not be the sole

inhibitory neurotransmitter at many of these synapses.

Mixed GABA-glycine synapses may mediate neurotrans-

mission at individual synapses in the spinal cord (174),

brain stem (194, 283, 325), and cerebellum (100). Interest-

ingly, GlyR activation appears to be able to inhibit

GABA A Rs via a phosphorylation-dependent mechanism

(229). This process may be important for regulating inhib-itory synaptic current magnitude at mixed GABA-glycine

synapses. There is evidence that the GABAergic compo-

nent of inhibitory neurotransmission at mixed synapses

may be upregulated in individuals suffering from heritable

disorders of glycinergic neurotransmission (see sect. VII).

Finally, presynaptic GlyRs have been functionally

characterized at calyceal synapses (373) and on terminals

synapsing onto rat spinal sensory neurons (172). Surpris-

ingly, in both cases GlyR activation is excitatory, leading

to increased neurotransmitter release.

2. Distribution of GlyR subunits

In situ hybridization was the first approach employed

to localize the distribution of individual GlyR subunits in

the rat. An advantage of this approach is its subtype

specificity, but a disadvantage is that transcript expres-

sion does not necessarily imply the surface expression of

functional receptors. Expression of ␣1-subunit mRNA in

adult rats was highest in the brain stem nuclei and spinal

cord, but it was also found in the superior and inferior

colliculi and in regions of the thalamus and hypothalamus

(245, 331). It was notably absent from cortical regions.

Thus, with few exceptions, its distribution is similar to

that of functional GlyRs as described above. In the rat,expression is detectable at embryonic day 15 and in-

creases to a maximum at around postnatal day 15, with-

out substantial changes in its distribution (245). Northern

blot analysis reveals that ␣1ins shares a similar distribu-

tion (244).

Prenatally, transcripts of the ␣2-subunit gene are

found throughout most of the central nervous system.

However, postnatally they decline sharply with little label

remaining by postnatal day 20 (9, 245, 392). Detectable

amounts of ␣2-transcripts do persist into adulthood, how-

ever, notably in the retina (see below), auditory brain

stem nuclei (293), and some higher brain regions (245).

The ␣2A-isoform is expressed more abundantly than ␣2B

during development, although the ␣2B-isoform is present

at higher levels in the adult (199).

The distribution and developmental changes in ␣3-

transcripts generally resemble those of ␣1-transcripts,

with the exception that ␣3-expression is much less in-tense at all developmental stages (245). As with the ␣1-

subunit, its expression intensity increases postnatally to

reach a maximum at around 3 wk (245). The ␣3L- and

␣3K-variants share similar distribution patterns (280).

GlyR ␣4-subunit transcripts are expressed at very low

levels (if at all) in the adult rat (293), although they are

strongly expressed in the spinal cord, dorsal root ganglia,

sympathetic ganglia, and the male genital ridge of the

chick (157).

GlyR ␤-subunit transcripts are distributed widely

throughout the embryonic and adult central nervous sys-

tem (125, 245). Although present at low levels prenatally,expression increases dramatically after birth and persists

into adulthood (245). The reason for this broad expres-

sion profile is somewhat puzzling given that these sub-

units do not form functional receptors in the absence of

␣-subunits.

3. Developmental switch from ␣ 2 to ␣1␤

Becker et al. (27) showed using protein expression

that fetal GlyRs are predominantly ␣2-homomers,

whereas adult receptors are predominantly ␣1␤-hetero-

mers. Such a switch is also supported by the mRNA

expression patterns described above. In the neonatal rat,the ␣1-, ␣2-, and ␤-subunits exist in abundance, implying

a mixture of receptor isoforms, but the switch towards

the adult isoform is complete by around postnatal day 20

(27, 121, 392). The sparse expression of the ␣3- and ␣4-

subunits suggests they may also be included in a minority

of adult GlyRs. Recent evidence suggests this switch may

not be as complete as originally thought and that ␣2-

subunit expression may remain at significant levels

throughout adulthood in the retina (see below) and audi-

tory brain stem (293). Although the mechanism responsi-

ble for triggering the developmental switch is not known,

it does not seem to require the activation of the GlyRsthemselves (247).

Given that ␣2-subunits alone are expressed in embry-

onic neurons, is it possible that homomeric ␣2-GlyRs may

mediate synaptic transmission? Takahashi et al. (361)

showed that the single-channel conductance and kinetic

properties of recombinant homomeric ␣2- and ␣1-GlyRs

were similar to those of native GlyRs in rat spinal neurons

at embryonic day 20 and postnatal day 22, respectively.

They also demonstrated an increased decay rate of the

glycinergic inhibitory postsynaptic currents (IPSCs) over

the same period that was consistent with the change in





channel kinetic properties (361). Subsequent studies have

supported these findings (12, 347). However, it is unlikely

that homomeric ␣2-GlyRs mediate inhibitory neurotrans-

mission for the following reasons. First, because ␤-sub-

units are required for GlyR postsynaptic clustering (188,

259), it is not certain how the ␣2-homomers would un-

dergo the prerequisite aggregation at postsynaptic densi-ties. Second, a recent study has found that ␣2-homomeric

GlyRs activate too slowly to effectively mediate synaptic

transmission (246). Given their wide distribution through-

out the nervous system during development, and the fact

that ClϪ fluxes are excitatory in developing neurons (114,

355), it seems more likely that homomeric ␣2-GlyRs me-

diate nonsynaptic cell-to-cell communication that could

be important for neuronal differentiation and synapto-

genesis (190). Glycinergic synapses in immature neurons

are probably comprised of ␣2␤-heteromeric GlyRs (219).

The single-channel conductance of synaptic GlyRs from

embryonic neurons is consistent with such a conclusion(12, 347).

4. A special case: the retina

This is considered separately because the profile of

GlyR subunit distribution is atypical and has been mapped

in detail and because a specific role for glycinergic syn-

apses has been proposed. GABA and glycine both func-

tion as inhibitory neurotransmitters in the retina (143,

176, 297, 390). In situ hybridization and immunohisto-

chemistry both show that GlyR ␣1-, ␣2-, ␣3-, and ␤-sub-

units have different patterns of distribution in the adult

rat (136, 146, 330). The ␣1-subunit is distributed predom-inantly on bipolar cells and on some ganglion cells in the

inner plexiform layer (136, 144, 145, 231). The ␣2-subunit

is distributed on amacrine cells and on almost all ganglion

cells, whereas the ␣3- and ␤-subunits are distributed

widely throughout the inner plexiform layer (136). A de-

tailed study in the mouse concluded that ␣1-subunits are

associated with synapses in the rod pathway between AII

amacrine cells and off-cone bipolar cells, whereas ␣3-

subunits are restricted to cone pathways (159). Together

these results indicate a spatial distribution of GlyR sub-

unit composition throughout the adult retina. Consistent

with this picture, Enz and Bormann (105) detected mRNA for all four GlyR subunits in RNA from whole retina, but

mRNA for only ␣1- and ␤-subunits in RNA isolated from

individual rod bipolar cells.

Electron microscopy has confirmed that the punctate

immunoreactivity seen with the light microscope is due to

clusters of GlyRs at the postsynaptic densities (146, 330).

Consistent with these anatomical studies, electrophysio-

logical investigations in the rat have revealed the pres-

ence of glycinergic inhibitory postsynaptic potentials

(IPSPs) in identified amacrine cells (119), ganglion cells

(153, 302, 368), and rod bipolar cells (77, 99, 303).

The synaptic distribution of GlyR subunits is spatially

distinct from that of GABA A R subunits, although individ-

ual ganglion cells may possess both types of synapse (195,

329). Recent studies have begun to address the possibility

that glycinergic and GABAergic transmission may have

distinct physiological roles. Although functional differ-

ences between GABAergic and glycinergic IPSPs in reti-nal neurons have been demonstrated (119, 302), the phys-

iological significance remains unknown. However, struc-

tural studies have provided evidence that the GABA and

glycine synaptic pathways participate in different func-

tional circuits. In particular, glycinergic synapses are

thought to play a specific role in the transmission of

dark-adjustment signals through the off-channel of the

rod pathway from amacrine cells to off-bipolar cells and

hence to off-ganglion cells (146, 330, 391). This circuit

contributes to the switch from day to night vision.

C. Distribution and Function in Other Tissues

1. Spermatozoa

The front of the mammalian sperm head contains a

large secretory vesicle termed the acrosome. The process

of fertilization is initiated when the sperm head contacts

the outer coat, or zona pellucida, of the egg. A zona

pellucida-specific glycoprotein, ZP3, forms the sperm re-

ceptor. Its interaction with sperm initiates a complex

intracellular signaling mechanism inside the sperm that

culminates in a calcium elevation that is thought to be

mediated at least partly by an influx through voltage-gated

calcium channels (115). This event, termed the acrosomereaction (AR), results in the release of acrosome hydro-

lytic enzymes by exocytosis. These enzymes induce vari-

ous protein modifications to ensure that the sperm re-

mains tightly bound to the zona pellucida while fusion

takes place between the sperm and egg plasma mem-

branes.

The activation of GlyRs and GABA A Rs in the sperm

plasma membrane appears to be essential for the AR

(257). There is considerable evidence that GlyRs exist in

sperm plasma membranes. For example, immunochemi-

cal studies have demonstrated the existence of GlyR ␣-

and ␤-subunit protein in porcine, mouse, and humansperm (49, 258, 332). An immunofluorescence study local-

ized the ␣-subunits to cell membranes in the periacroso-

mal regions of live mouse sperm (332). In addition, strych-

nine binding studies have revealed the presence of GlyRs

in hamster sperm (232).

Functional evidence for GlyR involvement has also

been demonstrated. For example, glycine initiated the AR

in a manner that was inhibited by strychnine or a GlyR

␣-subunit antibody (49, 332). Furthermore, studies using

fura 2-loaded human sperm showed that 50 nM strychnine

was also able to inhibit the ZP3-mediated calcium influx





(49). Finally, sperm from homozygous spasmodic and

spastic mice (which possess defective GlyR ␣1- and

␤-subunits, respectively) are deficient in their ability to

undergo the AR (333).

Thus the GlyR is likely to have a central role in the

AR. However, two questions remain about this process.

What is the concentration of ClϪ

inside sperm? Presum-ably, it must be high enough to force an outward (i.e.,

depolarizing) ClϪ flux upon GlyR activation. Second, what

is the glycine concentration in the oviduct where fertili-

zation takes place? Do the GlyRs remain tonically active

holding the sperm in a depolarized state preceding

the AR?

Harvey et al. (157) found that the ␣4-subunit gene is

expressed on the developing male genital ridge of the

chick and proposed that GlyRs containing this subunit

may contribute to the development of immature sper-

matogonia.

2. Endocrine pancreas

A pancreatic cell line, GK-P3, expresses functional

GlyRs. When activated, these receptors cause a depolar-

ization that increases the intracellular calcium concentra-

tion (393). A glycine receptor antibody displayed immu-

noreactivity with GK-P3 cells and with isolated rat pan-

creatic islet cells (393) prompting the authors to surmise

that GlyRs may also be expressed in islet cells in vivo.

However, there is as yet no electrophysiological evidence

for GlyRs in pancreatic islet cells.

3. Adrenomedullary chromaffin cells

High-af finity [3H]strychnine binding sites have been

shown to exist in catecholamine-secreting chromaf fin

cells of the adrenal medulla (415, 416). The same group

subsequently demonstrated that glycine can stimulate sig-

nificant catecholamine secretion from chromaf fin cells in

both in vitro and in vivo assays (414, 417). The presence

of GlyR ␣3-subunit mRNA (but not ␣1 or ␣2) was also

demonstrated by RT-PCR from RNA extracted from rat

adrenal glands. However, direct electrophysiological evi-

dence for glycine-activated currents in chromaf fin cells is

conspicuously absent to date.

4. Kupffer cells and other macrophages

A variety of pharmacological evidence, summarized

in Reference 184, suggests that GlyRs may at least par-

tially mediate the anti-inflammatory effects of glycine in

macrophages and leukocytes. Research on GlyR involve-

ment in these processes has focused on Kupffer cells,

which are specialized macrophages found in the liver.

Glycine has been shown to reduce the magnitude of li-

popolysaccharide-induced calcium transients in these

cells in a strychnine-dependent manner (122, 167). Similar

observations have also been made in neutrophils (398)

and hepatic parenchymal cells (304). Recent evidence

from Western blots, RT-PCR, and RNAse protection as-

says indeed suggest the presence of GlyR ␣1-, ␣2-, ␣4-,

and ␤-subunits in Kupffer cells (122).

5. Neural stem progenitor cells

Strychnine-sensitive glycine-gated currents are pres-

ent in postnatal, nestin-positive neural stem progenitor

cells (278). Consistent with this observation, RT-PCR and

immunocytochemical methods revealed the presence of

␣1-, ␣2-, and ␤-subunit RNA transcripts and ␣-subunit

protein, respectively.

III. STRUCTURE AND ASSEMBLY

A. General Structural Features

The nAChR is the most intensively investigated mem-

ber of the LGIC family. Consequently, most of the struc-

tural features of the GlyR have been deduced from its

homology with this receptor. LGIC receptors contain fi ve

subunits arranged pseudo-symmetrically around a central

ion-conducting pore. The membrane topologies of all

LGIC subunits are similar. This topology includes a large

NH2-terminal extracellular domain that contains the ago-

nist binding sites. A defining feature of LGIC subunits is

the conserved cysteine loop in this domain. All GlyR

subunits also harbor a second cysteine loop (309) that

incorporates a principal glycine-binding domain. As dis-

cussed in detail below, the crystal structure of acetylcho-

line-binding protein (AChBP) provides an excellent model

for understanding the structure of this domain (52).

Hydropathy analysis originally predicted an arrange-

ment of four ␣-helical transmembrane domains (TM1–TM4) per subunit. Although evidence exists for the inclu-

sion of ␤-sheet in the TM regions (134), the recent eluci-

dation of the crystal structure of the Torpedo nAChR TM

domains provides an overwhelming argument in favor of

the original four ␣-helical model (267). This structure,

determined by cryoelectron microscopy to a resolution of

4 Å by Miyazawa et al. (267), is a major advance in the

field. Finally, TM3 and TM4 are linked by a large, poorlyconserved, intracellular domain that contains phosphory-

lation sites and other sites for mediating interactions with

cytoplasmic factors. The structure and function of each of

these regions is now considered in detail.

B. Transmembrane Domains

1. Spatial organization

The principal role of the TM domains is to provide a

sealed barrier to separate the ion permeation pathway





from the apolar region of the lipid bilayer. In most ion

channels of known structure, this is achieved by a close

packing of amphipathic ␣-helices at angles close, but not

quite perpendicular, to the plane of the membrane (353).

This arrangement also applies to LGIC receptors, with

each subunit contributing an ␣-helical TM2 domain to the

lining of a single central water-filled pore. The TM1, TM3,and TM4 domains surround TM2 and provide the interface

with the lipid bilayer, thereby isolating TM2 from direct

contact with the surrounding hydrophobic environment.

Viewed from the synapse, TM1–TM4 are arranged consec-

utively in a clockwise manner, with TM1 and TM3 located

closest to TM2 (267). In the nAChR, the TM domains splay

outwards towards the extracellular membrane surface

and extend about two helical rotations (ϳ10 Å ) beyond

the hydrophobic membrane core. As noted by Miyazawa

et al. (267), the extracellular spaces between the splayed

helices appear to afford a lateral pathway (in addition to

the large central outer vestibule pathway) for ions toaccess the pore.

The remainder of this section attempts to relate the

Miyazawa TM domain structure with an abundance of

earlier information that also bears upon TM domain struc-

ture and function. However, before doing so, it is worth

briefl y considering three functionally based techniques

that have been of particular value in defining the second-

ary structure of ion channel pore-lining domains.

2. Methods for probing TM domain

secondary structure

A ) SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD. The sub-stituted cysteine accessibility method (SCAM) was ini-

tially applied as a means of identifying the secondary

structure of ion channel pore-lining domains (5, 8). The

method entails introducing cysteine residues one at a time

into the protein domain of interest. Cysteine reactivity is

then assayed by exposure to highly soluble, sulfhydryl-

specific reagents, generally methanethiosulfonate (MTS)

derivatives (180). If a functional property of the channel is

irreversibly modified upon exposure to such a reagent,

the cysteine is assumed to be exposed at the water-

accessible protein surface. If every second residue is

reactive, then the secondary structure is interpreted as␤-sheet (8), whereas if every third or fourth residue is

exposed, the structure is interpreted as ␣-helical (7). This

approach is now applied more widely to probe structural

changes in extramembranous domains (e.g., Ref. 234).

However, a drawback of applying this approach outside

the pore is that a lack of functional modification does not

necessarily mean that the residue has not reacted. In

other words, negative results cannot be interpreted. How-

ever, this limitation is less likely to apply in the spatially

restricted environment of an ion channel pore, where

attachment of a large side chain is more likely to affect

current flow, thus providing a generally more reliable

measure of cysteine reactivity. Various extensions to this

technique have also proven useful. For example, by de-

termining changes in cysteine reactivity in various func-

tional states (e.g., closed, open, and desensitized), it may

be possible to draw conclusions about state-dependent

structural changes. Similarly, by comparing state-depen-dent reaction rates of positively and negatively charged

reagents, it may be possible to estimate the local electro-

static potential (289, 405). The originators of SCAM have

provided an excellent review of its capabilities and limi-

tations (180).

B) TRYPTOPHAN SCANNING MUTAGENESIS. This approach in-

volves introducing tryptophan residues one at a time into

the domain of interest. Because tryptophan side chains

are bulky, it is assumed that if they protrude into the

relatively fluid lipid bilayer they should be less likely to

disrupt receptor structure and function than if they pro-

trude towards the protein interior (71). Experimentally,one or more basic functional properties (e.g., agonist

EC50) of each mutant receptor is measured, and then a

correlation is drawn between the position of the intro-

duced tryptophan and the severity of the functional con-

sequence. As with SCAM, any resulting periodicity is in-

terpreted as ␤-sheet or ␣-helix.

C) HYDROPHOBIC REAGENT REACTIVITY . In this approach,

employed extensively by Blanton and colleagues in the

nAChR (22, 40, 41), labeled hydrophobic reagents are

incubated with the receptor. The identity of the residues

that are covalently modified by these compounds is then

determined using biochemical assays. Residues thus iden-

tified are assumed to be exposed to the lipid bilayer. Again, any resulting periodicity is interpreted in terms of

secondary structure.

3. TM1

By connecting directly with the NH2-terminal do-

main, TM1 is ideally located to act as a linkage between

the ligand-binding site and the channel activation gate.

Hence, an unequivocal understanding of its structure and

relationship with TM2 is essential. The Torpedo nAChR

crystal structure identifies TM1 as an ␣-helix that is initi-

ated at the residue corresponding to Y222 of the ␣1-GlyRand enters the membrane at around M227. As stated

above, it is likely that water-filled space surrounds the

extracellular portion of this helix. In support of this, an

aqueous tyrosine-specific reagent labeled two tyrosines

(Y223, Y228) in TM1 of the ␣1-GlyR (222). Furthermore,

SCAM analysis on the nAChR revealed several extramem-

branous TM1 residues that are accessible to modification

by hydrophilic reagents (432). Several lines of evidence

implicate the extramembranous TM1 residues in LGIC

gating (e.g., Refs. 6, 38, 102, 363, 432). Throughout the

membrane-embedded portion of the nAChR TM1 there





appears to be a distinct absence of van der Waals contacts

with TM2, implying that a water-filled pocket separates

the respective domains (267). However, by homology

with nAChR, there may be a hydrophobic bond linking

I234 (or L237) and M12Ј in TM2 of the ␣1-GlyR. At its

intracellular end, the TM1 ␣-helix is probably terminated

by the aspartic acid at position 247.

4. TM2

Af finity labeling experiments employing pore-block-

ing substances first suggested an ␣-helical open state

structure of the nAChR TM2 (reviewed in Ref. 18). The

Torpedo nAChR structure of Miyazawa et al. (267) con-

firms the long-held view that this domain forms an ␣-helix

throughout its entire length (267). As summarized in Fig-

ure 1 A, an ␣-helical structure is also strongly supported

by SCAM analysis. Indeed, the luminal exposure patterns

as determined by SCAM and the Miyazawa structure are

entirely in agreement. SCAM also reveals a highly con-served pattern of residue exposure in the nAChR,

GABA A R, and 5HT3R (Fig. 1 A), suggesting that a similar

pattern applies to all LGIC members.

To facilitate comparison between different LGIC

members, a common TM2 numbering system is used

(265). This system assigns position 1Ј to the putative

cytoplasmic end of TM2 and 19Ј to the outermost residue

(Fig. 1 A). (Note that these assignments are confirmed by

the Miyazawa TM domain structure.) A complete SCAM

analysis of the GlyR TM2 is yet to be published. However,

experiments conducted to date indicate the following

GlyR ␣1-subunit residues line the pore: G2Ј, T6

Ј, R19

Ј, and A20Ј (234, 341). By homology with other LGICs (7, 234,

341, 409), the following residues are also likely to line the

pore: T7Ј, L9Ј, T10Ј, T13Ј, S16Ј and G17Ј (Fig. 1 A). When

viewed on an ␣-helical net, these residues form a hydro-

philic “strip” along one side of an otherwise hydrophobic

␣-helix (Fig. 1 B). A predicted cross-section through the

␣1-GlyR pore is shown in Figure 1C . The pore exposure

pattern of residues intracellular to G2Ј cannot currentlybe predicted for anionic LGICs because, as discussed in

detail in section IV B, they contain an additional proline at

position Ϫ2Ј that is likely to induce structural disruptions

around the internal pore boundary. Because the GlyR

␤-subunit TM2 has an unusually low sequence homology

with all other LGIC TM2 domains (Fig. 1 A), it will be of

interest to determine whether it also shares the consensus

residue exposure pattern.

Structural analysis shows TM2 to be kinked radially

inwards, attaining a minimum pore diameter at its mid-

point (267). Miyazawa et al. (267) propose that this con-

striction facilitates a tight hydrophobic coupling betweenTM2 residues of neighboring subunits at two levels in the

central region of the pore. The first contact is thought to

occur between L9Ј of one subunit and the 10Ј residue of the

adjacent subunit. In all GlyR ␣-subunits the 10Ј residue is a

threonine, but in the ␤-subunit it is a serine. The second

intersubunit contact occurs between residues homologous

to Q14Ј and T13Ј in neighboring TM2s of the GlyR ␣1-subunit

(or E14Ј and S13Ј in the ␤-subunit).

Although the 19Ј position defines the external border

of membrane-embedded portion TM2, the ␣-helical struc-

ture extends into extracellular space for another 2.5 turns

before terminating at the residue corresponding to V280

in the ␣1-GlyR (267). SCAM analyses on the GlyR and

FIG. 1. Pore-lining residues in the ␣1 glycine receptor

(GlyR). A: sequence alignments of the TM2 domains of indicated LGIC subunits. Positively charged residues areshaded in blue, and negatively charged residues are

shaded in yellow. Note that only cationic LGICs have a negatively charged residue at Ϫ1Ј. Circles denote pore-lining residues as identified by SCAM analysis (7, 234, 341,

409) or by cryoelectron microscopic analysis in the nico-tinic acetylcholine receptor (nAChR) (267). In the case of

the serotonin (5-HT3) receptor, dark circles denote thoseresidues identified as pore-lining by both Refs. 288 and316, whereas light circles denote residues identified as

pore-lining by Ref. 316 only. Additional GlyR ␣1-subunit

residues that are assumed by structural homology withother LGICs to line the pore are identified by squares. B:helical net representation of GlyR ␣1-residues with the

putative pore-lining residues denoted by a white back-ground. C : hypothetical cross-sectional view through the

␣1 GlyR pore. Pore-lining residues are indicated by the

white backgrounds. The exposure pattern of residuesdeeper than G2Ј cannot currently be modeled.





GABA A R confirm that most extramembranous TM2 resi-

dues have extensive contact with water (37, 234). In fact,

the SCAM analysis on the GABA A R even predicted an

␣-helical structure for these residues (37). The TM2-TM3

linker is formed by the ␣1-subunit residues, V280 to D284.

The roles of TM2 in forming the channel gate, in

controlling ionic selectivity, and in forming the bindingsites for agents of physiological and pharmacological im-

portance are considered in sections IV and VI.

5. TM3

According to the Miyazawa TM domain structure, the

␣1-GlyR TM3 ␣-helical domain starts at I285, with the

membrane-embedded portion extending from A288 to

H311 (267). There is strong support from functionally

based techniques that at least the external (NH2-terminal)

half of this domain forms an amphipathic ␣-helix. For

example, evidence from the nAChR using lipophilic

probes identified an ␣-helical like periodicity in the lipid-facing residues (40). A tryptophan scanning analysis iden-

tified an identical periodicity in the same set of residues

(76). In addition, SCAM analysis of the GABA A R using

water-soluble reagents also supported an ␣-helical peri-

odicity in the outer half of TM3 (400, 401). A satisfying

aspect of these studies was that the water-facing residues

were generally displaced by one from the lipid-facing

residues (see Ref. 223 for review). Consistent with struc-

tural predictions (267), the SCAM results strongly suggest

that this portion of the domain contributes to the lining of

a water-filled pocket distinct from the channel pore. Re-

cent SCAM studies on the GABA A R in the absence and presence of pharmacological agents suggest that this

pocket changes conformation as the channel gates (400 –402). An abundance of evidence from both the GABA A R

and GlyR, reviewed in section VI, provide a strong case

that residues in this pocket form binding sites for alcohols

and volatile anesthetics. The Miyazawa TM domain struc-

ture reveals that residues from TM3 are closely apposed

to residues from both TM2 and TM4 at several points

throughout their lengths (267). However, TM3 appears to

contact TM1 only towards the intracellular membrane

surface.

6. TM4

In addition to direct structural evidence (267), sev-

eral lines of functional evidence imply that this domain

forms an ␣-helix throughout its entire length. First, the

pattern of lipid-exposed residues is consistent with an

␣-helical periodicity as determined by both hydrophobic

probes in the nAChR (40, 41) and tryptophan scanning

mutagenesis in the GABA A R (171). Second, proteolytic

studies on GlyR ␣1-homomers did not identify cleavages

in membrane-associated fragments of this domain (221), a

result that is also consistent with an ␣-helical structure.

By structural homology with the nAChR, the ␣1-GlyR TM4

is likely to be initiated at K385 and terminated at V418,

with the intramembranous portion extending from K389

to I408 (267). Thus the ␣-helix extends about 2.5 turns

beyond the external membrane boundary. Although TM4

is closely apposed to TM3 throughout its length, its con-

tact TM1 appears confined to its intracellular half (267).

C. NH2-Terminal Ligand-Binding Domain

1. Structural homology with AChBP

The fresh water snail, Lymnaea stagnalis, produces

and stores a soluble AChBP in glial cells located near to

cholinergic synapses. When released by acetylcholine

stimulation, AChBP buffers the acetylcholine in the syn-

aptic cleft (350). This protein forms a stable homopen-

tamer and binds acetylcholine, ␦-tubocurarine, and ␣-bun-garotoxin with much the same af finity as does the ␣7-

homomeric nAChR (350). AChBP comprises 210 amino

acids and, although it lacks the TM domains, it provides a

full-length model of the NH2-terminal ligand-binding do-

main of LGICs. It also incorporates the signature cysteine

loop that is a unique feature of the LGIC family. It shares

a 20 –24% amino acid sequence homology with nAChR

subunits and a 17% homology with the GlyR ␣1 subunit

(Fig. 2). The crystal structure of this protein (52) repre-

sents a major breakthrough in our understanding of LGIC

structure and function. Due to both its significant se-

quence homology and to its functional similarity with the␣7-homomeric nAChR, its structure is considered an ac-

curate template of the NH2-terminal ligand-binding do-

main of the nAChR and, by inference, of other LGIC

members.

In three dimensions, AChBP forms a hollow cylinder

with an external diameter of 80 Å , a height of 62 Å , and an

inside diameter of 18 Å . Its size and general shape are in

good agreement with that previously determined from

electron diffraction images of Torpedo nAChRs (266). A

model of the GlyR ␣1-subunit ligand-binding domain

based on the AChBP structure is shown in Figure 3. Each

of the fi ve subunits is positioned in a radially symmetricalmanner around the central pore. When viewed from

above (i.e., from the synapse, looking towards the mem-

brane), the protein is said to resemble “a 5-bladed wind-

mill toy” (52). Individual subunits contain an ␣-helix near

the NH2-terminal extremity and then a series of 10

␤-sheets with short 310 helices following the second and

third ␤-sheets. The ␤-sheets 1–7 form a “twisted ␤-sand-

wich” with ␤-sheets 8 –10, resulting in 2 separate hydro-

phobic cores. Together the ␤-sheets form a modified im-

munoglobulin fold. Pockets are present at the subunit

interfaces, approximately midway between the top and





bottom of the protein, and abundant evidence (reviewed

in Refs. 74, 179) identifies these as ligand binding sites.

This pocket is lined by three loops from one subunit that

form the “ principal” (or ϩ) side of the ligand-binding

pocket, whereas three ␤-sheets from the adjacent subunit

form the “complementary” (or Ϫ) side of the pocket.

Viewed from the top of the complex, the complementary

side of each AChBP binding site is situated anticlockwise

relative to the principal side (52). The AChBP binding site

opens to the outside of the complex and, unlike the

Torpedo nAChR electron diffraction images (266), there is

no entry to the binding site from the central pore side of

the protein.

The AChBP structure reconciles many years of bio-

chemical and electrophysiological investigations into the

structure and function of the nAChR. As discussed later in

sections V and VI, it also reconciles an accumulation of

structure-function data from the GlyR. In particular, it

FIG. 2. Amino acid sequence align-ment of AChBP and the human ␣1 and ␤GlyR subunits. Secondary structural ele-

ments of AChBP and Torpedo nAChR areshown in gray, with ␤-strands repre-sented by arrows and helices represented

by cylinders (52, 267). Membrane-span-ning sections of the TM ␣-helices areshaded in dark gray. The NH

2-terminal

␣-helix is labeled by ␣, and two short polyproline helices are identified by 3

10.

Cross-linked cysteines are indicated by

the black brackets. The approximate lo-cations of the AChBP binding domainsare identified by red lines (and labeled

A– F , as appropriate) with known glycineand strychnine binding residues boxed inblack. The numbered assembly boxes are

outlined in green. Residues on the plusand minus sides of the interface areshaded yellow and blue, respectively.

Zinc-coordinating histidines are shown in pink. See Ref. 276 for justification of thesequence alignment used in the zinc-co-

ordinating region. Putative glycosylationsites are shaded in green, and putative

phosphorylation sites are shaded in red

and labeled accordingly. A region of theTM3-TM4 domain involved in determin-

ing single-channel conductance in5-HT3Rs is shaded in gray. The TM3 in-sertion domain, the gephyrin binding do-main, and SH

3homology domains are

boxed and labeled in light blue, darkblue, and orange, respectively.





provides an excellent basis for understanding the glycine

and strychnine binding sites and the zinc inhibitory site.

It was recently proposed that sections of the ␣1-GlyR

NH2-terminal domain between residues 158 –165 and 181–191 may be associated with the plasma membrane (223).

Because the AChBP domain corresponding to 158 –165 is

located at a subunit interface well away from the mem-

brane, a direct membrane interaction seems unlikely.

However, residues 181–191 indeed lie toward the lowest

point of the structure, between ␤-sheets 8 and 9, and thus

could conceivably dip into the membrane.

Apart from the direct polypeptide chain linkage be-

tween ␤-sheet 10 and TM1, structural and functional evi-

dence suggest 2 likely points of contact between the

ligand-binding domain and the transmembrane domain.

These regions are the conserved cysteine loop and the

loop linking ␤-sheets 1 and 2 of AChBP. This later loop is

also known as “loop 2.” Both loops have been proposed to

FIG. 3. Homology model of the ␣1 GlyRligand-binding domain. A: backbone ribbon

representation of the ␣1 GlyR viewed alongthe 5-fold axis of symmetry from the synapticcleft. The sequence alignment used to gener-

ate this model is shown in Fig. 2. Differentcolors indicate different subunits. Dotted lines

mark the subunit interfaces with ϩ / Ϫ signsindicating the subunit faces that contribute tothe interface. The putative inhibitory zinc-binding region is shown in blue with H107 and

H109 side-chains shown as bonds in pink.[Model from Nevin et al. (276).] B: stereo viewof the inhibitory zinc-binding site (as proposed

in Ref. 276) viewed from within the vestibulelumen along the direction of the arrow in A.Only two subunits are shown for clarity. Side-

chains of H107 and H109 from the ϩ face( right) and H107, H109, E110, and T112 fromthe Ϫ face (left) are shown as bonds, with

standard coloring according to atom. The pinksphere indicates the location of bound zinc.(Image courtesy of Dr. Brett Cromer and Prof.

Michael Parker.)





interact closely with the TM2-TM3 linker domain (181,

267), and the nature of these proposed interactions is

considered in more detail in section V D.

2. Glycosylation

As shown in Figure 2, the GlyR ␣1-subunit contains a glycosylation consensus site at N38, with other ␣-subunits

containing similar sites at the homologous positions. The

GlyR ␣2-subunit contains additional consensus sites at

N45 and N76 (199). On the other hand, the ␤-subunit

contains consensus sites at N33 and N220 (137). The first

suggestion that the ␣1-subunit may be glycosylated was

the finding that mutations to N38 prevented surface ex-

pression of functional ␣1-GlyRs (10, 200). Recently, it was

found that glycosylation of ␣1-subunits is a necessary

prerequisite for homomeric receptor assembly and that

receptor assembly is required for transit from the endo-

plasmic reticulum to the Golgi apparatus and subse-

quently to the cell membrane (140). The question of whether ␤-subunits are glycosylated remains to be ad-

dressed.

D. Large Intracellular Domain

As the large TM3-TM4 domain is poorly conserved

among LGIC members, both in terms of its length and

amino acid sequence, it is likely to exhibit considerable

structural variation as well. The only structural informa-

tion to date suggests that the Torpedo nAChR intracellular

domains form a hanging gondola-type structure withtransverse holes (or “ portals”) connecting the pore with

the cytoplasm (266). Because these portals are approxi-

mately the same size as a permeating ion plus its first

hydration shell (266), they are ideally suited to influence

ion permeation. Indeed, it has recently been shown that

the deletion of three positively charged residues in the

5-HT3A R TM3-TM4 domain dramatically increases the

pore unitary cation conductance (183), implying that

these residues may frame the portals. The homologous

region of the GlyR ␣1-subunit is denoted by gray shading

in Figure 2. The GlyR ␤-subunit has an unusually large

internal domain, comprising 130 residues, whereas the

␣1-subunit contains 86 residues (Fig. 2). The intracellular domains of both ␣- and ␤-subunits contain a variety of

sites that mediate interactions between the GlyR and

cytoplasmic factors. These putative interaction sites are

now considered.

1. Ubiquitination domain

Under appropriate conditions, intracellular ubiquitin

molecules can covalently attach themselves to specific

lysine side chains on the cytoplasmic protein surface. In

fact, multiple ubiquitin molecules can attach themselves

end to end in a piggyback manner, resulting in a condition

termed “ polyubiquitination.” Ubiquitination or polyubiq-

uitination precipitates the internalization and degradation

of many protein types, including surface-expressed ␣1-

GlyRs (55). Following internalization, the ubiquitin mole-

cules induce the 49-kDa GlyR ␣1-subunit to be proteolyti-

cally nicked into a glycosylated (i.e., NH2-terminal) 35-kDa fragment and a 17-kDa COOH-terminal fragment.

These fragment sizes are consistent with the ubiquitina-

tion domain lying in the large intracellular domain. The

TM3-TM4 domain contains a total of 10 lysine residues

(Fig. 2), several of which probably need to be individually

ubiquitinated before GlyRs can be endocytosed (55). This

mechanism is likely to be important in regulating the

number of surface-expressed GlyRs per postsynaptic den-

sity.

2. SH 3

-binding motif

Because prolines induce kinks into peptide chains,regular spacing of these residues can form helical struc-

tures known as polyproline (PII) helices. Circular dichro-

ism studies reveal the GlyR ␣1-subunit to contain a sig-

nificant fraction (9%) of this structure (58). A certain class

of protein-protein interaction sites, termed SH3 domains,

are formed from PII helices (290). As recently noted (58),

GlyR ␣1- and ␤-subunits both contain SH3 consensus

sequences in their large intracellular domains (Fig. 2).

Although the role of these domains has yet to be investi-

gated, they may be involved in GlyR traf ficking or cy-

toskeletal attachment.

3. Phosphorylation sites

The locations of phosphorylation consensus sites in

the ␣1- and ␤-subunits are shown in Figure 2. The evi-

dence that phosphorylation of these sites is able to mod-

ulate GlyR function is considered in section VII A.

4. Gephyrin binding domain

This important molecule has long been known to

copurify with the native GlyR as a 93-kDa protein (300).

Kirsch and Betz (188) showed that it mediates the clus-

tering of GlyRs at postsynaptic sites. Gephyrin interactswith a large and growing number of binding partners,

suggestive of a high degree of complexity in the regulation

of GlyR clustering. A description of the interactions of

gephyrin with molecules other than the GlyR is beyond

the scope of this review. Developments in this area are

moving rapidly, and recent progress has been covered in

several excellent reviews (189, 190, 219). The GlyR gephy-

rin contact site was isolated to an 18-amino acid domain

in the central region of the ␤-subunit TM3-TM4 loop (259)

(Fig. 2). Insertion of the gephyrin binding domain into the

␣1-subunit promotes the clustering of ␣1-homomeric





GlyRs (220, 259). A site-directed mutagenesis study iso-

lated gephyrin binding activity to multiple hydrophobic

residues in this domain (191). A hydropathy plot suggests

that this region forms an irregular amphipathic helix, with

the putative gephyrin-binding residues located along the

hydrophobic side.

5. Basic cluster required for TM3 integration

A positively charged cluster, RFRRKRR, located

close to the intracellular boundary of TM3 in the ␣1-

subunit (Fig. 2), appears to be important for the correct

membrane insertion of TM3. It was found that neutraliza-

tion of positive charges in this cluster prevented the cor-

rect translocation of TM3-TM4 into the lumen of the en-

doplasmic reticulum (328). This effect was rectified by

deleting positive charges from TM2-TM3. From these re-

sults, it was concluded that the basic cluster is necessary

to compensate for the positively charged residues in TM2-

TM3 which would otherwise preclude the correct mem-

brane insertion of TM3 (328).

E. Receptor Assembly

1. Subunit stoichiometry and arrangement

The subunit composition of the GlyR was determined

by cross-linking its polypeptides with cross-linking re-

agents of various specificities and lengths (209). Because

the size of the largest cross-linked product totaled ap-

proximately fi ve times the mean individual subunit size,

functional membrane GlyRs were concluded to comprise pentamers. Of course, since a pentameric subunit ar-

rangement was well-established in other LGIC members

(and now in AChBP), it is scarcely conceivable that the

GlyR quaternary structure would have been different.

Oddly, however, evidence for a trimeric ␣1-homomeric

subunit composition has recently been deduced on the

basis of both laser scattering and single particle electron

microscopic analyses (413). A substantial measure of

credibility must be granted to this study as the same

group also proposed a pentameric GABA A R structure us-

ing the same techniques. These are the only GlyR images

published to date, and further investigation into the basisof these findings appears warranted. One possibility is

that the structure is distorted by a closely associated

protein.

Although GlyRs almost certainly comprise pentam-

eric subunit complexes, the stoichiometry and subunit

arrangement of heteromeric receptors are less certain. An

invariant 3␣:2␤ stoichiometry was proposed based on the

observation that ␣-subunits predominated over ␤-sub-

units in all tissues examined (209). Although no more

direct evidence in favor of any particular stoichiometry

has ever been presented, the 3␣:2␤ ratio is a long-held

dogma in the field. Even if this stoichiometry is correct,

there is no compelling rationale for distinguishing a side-

by-side ␤-subunit arrangement from one whereby the

␤-subunits are separated by an ␣-subunit. Knowledge of

this is particularly important for understanding GlyR mo-

lecular pharmacology because binding sites of all types

are located at subunit interfaces (74), and the number of ␣-␣, ␣-␤, ␤-␣, and ␤-␤ interfaces per receptor cannot

currently be determined. A careful reanalysis of subunit

stoichiometry and arrangement in ␣␤-heteromeric GlyRs

is overdue.

2. Intersubunit contact points and assembly domains

GlyR ␣1- and ␣2-subunits appear to be able to coas-

semble in a random binomial manner that is dependent

only on the relative abundance of each subunit (200).

However, when ␤-subunits are present, the ␣:␤ subunit

stoichiometry appears to be invariant, as inferred from

the monotonic nature of the glycine dose-response (200).The regions of the GlyR ␤-subunit responsible for this

behavior have been investigated in detail (140, 200). The

initial characterization involved coexpressing ␣1-subunits

with chimeric subunits made from ␣1- and ␤-subunits.

Fixed assembly was found to require the extracellular,

but not the TM or intracellular regions, of the ␤-subunit

(200). To further delineate the regions responsible for

subunit assembly, certain divergent domains (termed “as-

sembly boxes”) in the ␤-subunit NH2-terminal domain

were mutated back towards the ␣1-subunit sequences to

see whether they permitted the individual chimeric sub-

units to express as homomers. Indeed, the introduction of various combinations of boxes permitted homomeric as-

sembly (200). The important boxes involved in this pro-

cess are shown in Figure 2. As a minimum requirement,

box 1 has to combine with either box 3 or box 2 plus box

8 to result in ␣1-homomer formation (140, 200). The crit-

ical ␣1-subunit residues that must be set towards the

␣1-subunit sequence are as follows: box 1 (P35, N38, S40)

and box 3 (L90, S92), or box 2 (P79) and box 8 (N125,

Y128) (140). Note that N38 is a glycosylation site. The

intersubunit contact points between AChBP subunits and

their predicted counterparts in the GlyR ␣1-subunit are

shown in Figure 2. It is apparent that the boxes do not

directly form the interfaces, although boxes 3 and 8 lie

directly adjacent to interface sites. This suggests that the

box mutations act allosterically to modulate the interface

conformation. The roles of the interface residues them-

selves in controlling subunit assembly have yet to be

investigated.

3. Effects of high receptor density

The injection of increasing amounts of ␣1-subunit

cDNA into Xenopus oocytes results in a progressive in-

crease in both maximum current ( I max) and glycine sen-





sitivity (90, 362). A recent study has provided a possible

explanation for this. When ␣1-GlyRs containing intro-

duced gephyrin binding domains were clustered using

gephyrin, they exhibited an additional extremely fast de-

sensitizing current component (220). Although the glycine

EC50 of the peak current was similar to that of unclus-

tered receptors, the EC50 of the plateau current was sig-nificantly reduced. Fast (submillisecond) solution appli-

cation to small (HEK293) cells was required to see the fast

desensitizing component. Since such rapid solution ex-

change is dif ficult to achieve with Xenopus oocytes, it

seems possible that the fast desensitizing component was

missed in the earlier study. These effects could be the

result of direct interactions between adjacent receptors,

allosteric actions of gephyrin on the ␣-subunit, or deple-

tion of a cytoplasmic cofactor necessary for normal re-

ceptor function.

4. Coassembly with other LGIC subunitsFunctional evidence suggests that the GABA C 1-

subunit can coassemble with glycine ␣1- and ␣2-subunits

in vitro (287). This study showed that the recombinant

expression of a mutant 1-subunit with the ␣1- or ␣2-

subunits caused a change in the gating of GABA-induced

currents. Because homomeric GlyRs were not activated

by GABA, it was proposed that the change in pharmacol-

ogy must have been due to coassembly of 1- with ␣-GlyR

subunits. This finding raises the intriguing possibility that

such heteromeric receptors might also exist in vivo.

IV. STRUCTURE AND FUNCTION OF THE PORE

A. Functional Properties of the Pore

1. Ionic selectivity

Although GlyRs are strongly selective for anions over

cations, they have a small but measurable permeability to

K ϩ and Na ϩ (45, 186). Native GlyRs expressed in cultured

neurons have a permeability sequence of SCNϪϾ NO3

ϪϾ

IϪ Ͼ Br Ϫ Ͼ ClϪ Ͼ FϪ (45, 107). This sequence is in

proportion to the ionic hydration energies, implying that

the removal of waters of hydration is the major barrier toion channel entry. Because electrostatic interactions with

pore sites are, by inference, less important, this sequence

corresponds to a “weak field strength” binding site. The

GlyR pore also exhibits anomalous mole-fraction behav-

ior, suggesting the presence of at least two interacting

binding sites (45, 107). The permeability has also been

probed with large organic anions. From space-filling mod-

els of the molecules tested, the narrowest part of the pore

is estimated to have a diameter of at least 5.2 Å in spinal

neuron GlyRs (45), 5.5– 6.0 Å in hippocampal neuron

GlyRs (107) and 5.22–5.45 Å in recombinant GlyRs (324).

By comparison, cationic LGICs also possess a weak field

strength binding site, but they have not been shown to

display anomalous mole fraction effects and have a sig-

nificantly larger minimum pore diameter of at least 7.5 Å (225).

2. Single-channel conductance

GlyRs display multiple unitary conductance states

(45, 46). A useful comparison of GlyR conductance states

in native neuronal GlyRs, as well as in various recombi-

nant subunit configurations, is presented in Table 1 of

Reference 307. Recombinant ␣1-GlyRs exhibit fi ve con-

ductance states ranging between 20 and 90 pS, with the

90-pS state occurring with the greatest frequency. The ␣2-

and ␣3-GlyRs share the same conductance states but also

exhibit a frequently visited 110-pS conductance level. This

characteristic is conferred to the ␣1-GlyR by the G2Ј A

(␣1 3 ␣2) mutation (46). Coexpression of ␣1-subunits

with the ␤-subunit eliminates the highest conducting lev-els, leaving a 45-pS state as the most frequently occurring

state (46). Incorporation of the E20ЈS (␤ 3 ␣1) mutation

into the ␤-subunit confers ␣1-GlyR-like conductance lev-

els to heteromeric GlyRs. Because neither mutation abol-

ishes the preexisting conductance states, it is dif ficult to

determine whether their actions are mediated allosteri-

cally or via direct interactions with permeating ions. The

relative probabilities of entering the predominant con-

ductance states do not vary with agonist concentration

(25, 374).

As mentioned above, elimination of a series of three

positively charged residues in the 5-HT3A R TM3-TM4 do-main resulted in a dramatic increase in the single-channel

conductance (183). It was proposed these residues might

line a narrow portal that links the pore with the cytosol. It

is yet to be established whether the homologous residues

in the GlyR ␣- or ␤-subunits influence the unitary chloride

conductance. However, since both positively and nega-

tively charged residues line the corresponding domain in

the GlyR ␣1-subunit (Fig. 2), and the precise alignment is

unknown, it is dif ficult to predict what this influence

might be.

B. Molecular Determinants of Ion Selectivity and Conductance

Much of the groundwork for our current understand-

ing of GlyR permeation mechanisms has come from stud-

ies on the nAChR. One classic experiment showed that

the open-channel blocker QX-222 reached as far as the 6Ј

position when applied externally in the open state (61,

224), implying that the most constricted section of the

pore was located more internally than this point. Because

residues in the narrowest part of the pore are more likely

to influence both unitary conductance and ion selectivity,





this in turn implied that the residues near the intracellular

boundary of TM2 controlled both processes. A recent

SCAM study has provided the most definitive functional

evidence to date for a drastic pore constriction between

the Ϫ2Ј to the ϩ2Ј positions (404). It should be noted,

however, that the Miyazawa TM domain structure shows

the pore widening considerably from the ϩ2Ј to Ϫ2Ј positions (267). The reason for this apparent mismatch is

not yet understood.

Charged residues are obvious candidates as ionic

selectivity sites. As shown in Figure 1 A, the nAChR ␣1-

subunit contains four charged residues in the vicinity of

TM2: a negatively charged aspartic acid at Ϫ4Ј (the “cy-

toplasmic ring”), negatively charged glutamic acids at Ϫ1Ј

(the “intermediate ring”), and 20Ј (the “outer ring”) and a

positively charged lysine at 0Ј. Three lines of evidence

indicate that K0Ј faces away from the pore: 1) charge-

reversal mutations have no effect on single-channel con-

ductance (168), 2) the pore has a strong negative electro-static potential in this region (289), and 3) the Miyazawa

TM domain structure unequivocally shows K Ј0 facing

away from the pore (267). Although charge-reversal mu-

tations to the cytoplasmic and outer rings significantly

affect single-channel conductance (168, 169), neither site

appears to significantly impede the access of negatively

charged molecules to deeper regions of the pore (404).

However, mutations to the intermediate ring strongly in-

fluence both single-channel conductance (168) and pore

effective diameter (387). In addition, mutations to 2Ј res-

idues also affect cation selectivity and effective diameter

(reviewed in Ref. 225). Thus residues at the Ϫ1Ј and ϩ2Ј

positions, which are separated by one ␣-helical turn in thenarrowest part of the pore, form the main selectivity filter.

Structural predictions indicate that both side chains have

extensive exposure to the pore (267).

By comparing TM2 sequences between the ␣7-

nAChR and the ␣1-GlyR, Galzi et al. (126) sought to

determine the minimum number of GlyR residues re-

quired to switch the nAChR pore from cation selective to

anion selective. The minimum requirement was found to

be the E-1Ј A and V13ЈT mutations as well as the insertion

of a proline between Ϫ2Ј and Ϫ1Ј (i.e., Ϫ2ЈP). The same

mutations achieved a similar effect in the 5-HT3R (149).

Because the E-1Ј A and V13ЈT mutations alone do notconvert selectivity, they are considered to be “ permissive”rather than “essential” for anion permeability (73). Inter-

estingly, the proline insertion site was not critical: selec-

tivity reversal was also effected by inserting it into the

Ϫ4Ј position (73). Together these results imply that selec-

tivity reversal required a change in either the lumen ge-

ometry or in the exposure profile of side chains lining the

selectivity filter.

The reverse triple mutation (A-1ЈE, T13Ј V and dele-

tion of Ϫ2ЈP) was subsequently found to convert ␣1-GlyR

selectivity from anionic to cationic (185). However, the

resultant channels had a low conductance (3 pS at Ϫ60

mV) and converted the ratio of permeabilities ( P Cl / P

Na )

from 24 to 0.3, implying that ClϪ permeation may have

been reduced without a concomitant increase in Na ϩ

permeation. A stronger case for an increase in cation

permeability was made by the same group when they

showed that the reverse double mutation (A-1ЈE and de-letion of Ϫ2ЈP) decreased the P

Cl / P Na further to 0.13,

while increasing the unitary cation conductance to 7 pS at

Ϫ60 mV (186, 272). Furthermore, these mutations in-

creased the effective pore diameter of this GlyR to ap-

proximately the value seen in the nAChR. Curiously, how-

ever, selectivity reversal was also achieved by incorporat-

ing only the A-1ЈE mutation into both the ␣1-GlyR and the

1-GABA A R (186, 406). From this result, it is tempting to

speculate that anion selectivity is associated with a net

positive electrostatic charge caused by both the elimina-

tion of the negative charge at E-1Ј and an enhanced pore

exposure of R0Ј.However, one problem with the “net positive charge”

model of anion permeation is that the anion selectivity of

the triple mutant ␣7-nAChR is not affected by the charge-

eliminating K0ЈQ mutation (73). In addition, the E-1Ј A

mutation alone did not increase anion permeability (73).

Indeed, these two observations prompted Corringer et al.

(73) to suggest that the anion-selective nAChR pore may

be lined by polar groups from the peptide backbone.

Further insight into this issue was recently provided by

the finding that the 1-GABA A R anion-to-cation selectivity

switch was conferred by the charge-reversing mutation

R0ЈE, but not by the charge-eliminating mutations R0ЈC or R0ЈM (406). It appears feasible to reconcile all these

findings by proposing that a net negative charge in the Ϫ1Ј

region is associated with cation selectivity, whereas a net

neutral or positive charge is associated with anion selec-

tivity. In the event of a neutral potential in this region,

positive dipoles of local polar groups may confer anion

permeability. A critical test of this proposal would be to

quantitate the GlyR pore electrostatic potential profile

using SCAM (289, 405). It would also be informative to

investigate the effects of a variety of substitutions at the

Ϫ1Ј and 0Ј positions to differentiate the effects of side

chain charge, size, hydrophilicity, and hydrophobicity onion selectivity and pore effective diameter. Unfortunately,

however, mutations to these residues have a habit of

precluding functional receptor expression.

The influence of ␤-subunits on ion permeation has

yet to be investigated in detail. As shown in Figure 1 A, ␣-

and ␤-subunits have a low sequence homology through-

out TM2. Of particular note, the ␤-subunit includes an

alanine-for-proline substitution at Ϫ2Ј and a proline-for-

glycine substitution at position 2Ј. These substitutions

suggest the ␤-subunit secondary structure may be differ-

ent in the pore selectivity filter. However, as summarized





above, experiments to date suggest the ␤-subunit does not

induce drastic changes in permeation characteristics.

The effect on permeation of the outer ring of charge,

formed by R19Ј, has also been investigated in the ␣1-GlyR.

Elimination of this charge by the human startle disease

mutations R19ЈL and R19ЈQ caused a decrease in the

unitary ClϪ

conductance (208, 305). More recently, it wasshown that the R19Ј A and R19ЈE mutations increased the

unitary cation conductance in cation-selective GlyRs and

changed the rectification properties of the pore (272).

Together, these results are consistent with an electro-

static contribution of R19Ј to ion permeation. However,

R19Ј does not preclude the entry of positively charged

molecules into the GlyR pore (341, 343) and is more likely

to affect conductance by concentrating anions in the

outer vestibule (186). The ␤-subunit E20ЈS mutation,

which neutralizes a negative charge at the adjacent posi-

tion, may also increase the single-channel conductance

via an electrostatic mechanism (46). An impressive array of ␣1-GlyR pore functional prop-

erties was reconciled by a three-dimensional Brownian

dynamics simulation study (284). This study used a model

of the pore based on the TM2 primary structure and the

best available estimates of pore diameter prior to publi-

cation of the Miyazawa study (267). The selectivity filter

diameter was permitted to vary between 6 and 8.3 Å , but

the charge at R0Ј was held constant at ϩ0.375e. Under

these conditions, the first ClϪ experiences a deep energy

minimum near R0Ј. A second ClϪ entering the pore expe-

riences a weaker energy minimum near the pore mid-

point. These two ions exist in equilibrium. A third entering

ClϪ abolishes these energy wells, allowing the innermostion to escape into the intracellular solution. In light of the

preceding discussion, it would be interesting to determine

the effects on permeation of variations to the charge at R0Ј.

C. The Channel Activation Gate

This gate refers to the physical barrier that stops ions

from traversing the pore in the unliganded state. There is

no information available to date as to its location in the

GlyR. There are, however, two schools of thought as to its

location in other LGIC members. One proposal, supported

by the structural analyses of Unwin, Miyazawa, and col-leagues (266, 267, 376), is that the TM2 domains are

kinked inwards to form a centrally located gate near the

highly conserved L9Ј residue. However, the results of

SCAM experiments on the GABA A R and nAChR are in-

consistent with the gate lying more externally than the 2Ј

position (7, 289, 409). A particularly detailed study on the

nAChR, which probed the accessibility of cysteines intro-

duced into TM2 to both sides of the membrane in the

closed and open states, delimited the gate to the same

narrow pore region (Ϫ2Ј to ϩ2Ј) that houses the selectiv-

ity filter (404).

D. Molecular Mechanisms of Desensitization

Desensitization is a property whereby an agonist-

gated channel closes in the continued presence of agonist.

In general, the rates of onset and recovery from desensi-

tization are important parameters governing the size, de-

cay rate, and frequency of fast synaptic currents (175).Until recently, it was considered that GlyRs desensitized

too slowly to influence these parameters (219). However,

a recent study using ultra-fast solution exchange identi-

fied a very rapid desensitization component (decay time

constantϳ5 ms) that is induced by either the clustering of

␣1-GlyRs (220) or by coexpression of the ␣1- with the

␤-subunit (268). This rapid component could conceivably

influence the properties of repetitive glycinergic synaptic

currents. However, further research is required to clarify

the role, if any, of GlyR desensitization at glycinergic

synapses.

The location of the desensitization gate in the GlyR isnot yet known. However, SCAM evidence from the

nAChR suggests that the desensitized state is associated

with a crimping of the channel pore between the Ϫ2Ј and

9Ј residues (403). In contrast, a similar approach showed

that the activation gate crimps the pore between only the

Ϫ2Ј and ϩ2Ј positions.

A growing body of evidence reveals that intracellular

domains can profoundly influence GlyR desensitization. A

functional comparison of human ␣3K- and ␣3L-GlyRs

showed that the removal of a 15-amino acid segment from

the large intracellular domain dramatically increased the

desensitization rate (280). Desensitization rate is also dra-

matically enhanced by the human startle disease muta-tions, I244N and P250T (also known as P-2ЈT), in the

␣1-GlyR TM1-TM2 loop (237, 334). Other mutations in this

region, including W243A, I244A, and A251E (also known

as A-1ЈE), have similar effects (186, 237). The relationship

between desensitization rate and the properties of side

chains introduced into the P250 position showed that

bulky hydrophobic residues yielded the fastest desensiti-

zation rates (51). Thus desensitization rate is exquisitely

sensitive to structural perturbations in the TM1-TM2 loop.

However, in the GlyR at least, such observations are of

phenomenological interest only until it can be demon-

strated that events that alter desensitization in vivo do soby varying the conformation of this domain.

V. AGONIST BINDING AND

RECEPTOR ACTIVATION

A. Introduction

This section considers the molecular basis by which

agonists bind to and activate (or gate) the GlyR. In par-

ticular, it will consider the following important questions.





What is the structure of the binding site and where is it

located? What is the molecular basis for binding site

selectivity? What structural changes underlie the activa-

tion of the receptor? One problem with addressing these

questions is that it is dif ficult to experimentally dissect

binding from gating mechanisms as the two processes are

tightly coupled (72). To understand how this separationmay be achieved, it is necessary to briefl y consider the

theory of receptor activation.

1. Review of basic receptor theory

In its simplest form, the receptor activation process

can be represented as:

A ϩ R ¢ O ¡

K A

AR ¢ O ¡

E

AR*

where A is the agonist, R is the vacant receptor, AR is

occupied but shut, and AR* is occupied and activated. The

equilibrium constant for binding (or binding af finity) is

given by:

K A ϭ A R / AR ϭ koff / kon (1)

where koff is the dissociation rate constant (units of sϪ1)

and kon is the association rate constant (units of MϪ1⅐sϪ1).

The equilibrium constant for gating (or ef ficacy) is given

by:

E ϭ AR* / AR

ϭ

␤ / ␣ ( 2)

where ␤ is the opening rate constant and ␣ is the closing

rate constant (both with units of sϪ1). With the use of

classical receptor theory (along with some simplifying

assumptions), it can be shown (72) that the agonist con-

centration required for a half-maximal response:

EC 50 ϭ K A / 1ϩ E (3)

and the maximum fraction of receptors in the activated

state (or maximum open probability)

P omaxϭ E / 1ϩ E ( 4)

Equation 3 tells us that the EC50 is a function of both

the binding and gating properties of the receptor. Equa-

tion 4 tells us that variations in E have no measurable

effect on maximum open probability unless they occur

within a limited range of ϳ0.1–10.

The structural basis of GlyR binding and gating has

mainly been investigated using site-directed mutagene-

sis coupled with functional (i.e., electrophysiological)

analysis. If a mutation affects mainly K A , then it is

assumed to affect binding, either directly or allosteri-

cally. If a mutation affects mainly E , then the mutated

residue is assumed to affect the conformational change

leading to the open state, either directly or allosteri-

cally. Single-channel kinetic analysis can provide rea-

sonably direct estimates of both K A and E . A more

convenient, but less precise, method involves measuringrelative changes in the peak whole cell current ( I

max) acti-

vated by partial agonists. Since I maxϭ inP omax, where i is

single-channel conductance and n is number of activated

receptors, I max provides a qualitative measure of changes in

E , assuming that i, n, and desensitization rate remain con-

stant.

2. Concerted versus sequential models

of receptor activation

The above model of receptor activation is an over-

simplification because ␣-GlyRs, as pentameric multim-

ers, contain fi ve agonist binding sites. Two starkly con-trasting models have been proposed to describe multi-

subunit protein allosteric mechanisms. These are the

coupled Monod-Wyman-Changeux (MWC) model (60)

and the uncoupled or sequential Koshland-Nemethy-

Filmer (KNF) model (193). In the simplest version of

the MWC model, all the subunits change conformation

simultaneously, and in consequence, the receptor can

exist in only the closed or entirely activated states. In

contrast, in the KNF model, each subunit can indepen-

dently adopt a specific conformation change depending

on the number of bound agonist molecules, leading to a

series of intermediate protein conformational states.Extended MWC models that allow for multiple func-

tional states and subunit asymmetry can explain many

characteristics of nAChR behavior (60). In particular,

two simple observations that support an MWC model

over a KNF model are that 1) nAChR single-channel

conductance is independent of agonist concentration

and 2) spontaneous channel openings are occasionally

seen in the absence of agonist. As discussed in the

ensuing paragraphs, the information available to date

also favors an MWC model for the GlyR.

B. Kinetic Models of Glycine Receptor Gating

The single-channel kinetic properties of GlyRs were

first characterized in native receptors from embryonic

mouse spinal neurons (374). This study revealed several

important features of GlyR kinetic behavior. First, unlike

nAChRs, GlyR channel openings did not occur in the

absence of agonist. Second, although the single-channel

conductance was not constant, it displayed no variation

with agonist concentration. The third and perhaps most

revealing feature was that there were at least three expo-

nential components in the open period distributions.





Short-lived channel bursts (corresponding to the faster

exponential components) predominated at low concen-

trations, whereas the relative frequency of longer lived

bursts (slower exponential components) increased at

higher concentrations. It was therefore concluded that

progressively longer-lived states were associated with in-

creasing numbers of bound glycine molecules and thatmaximal channel activation therefore required a mini-

mum of three bound glycines (374). Because the molec-

ular identity of the mouse GlyRs is not known, the num-

ber of glycine binding sites per receptor is uncertain.

Irrespective of this, the three-open-state model is in

broad agreement with several more recent studies on

recombinant ␣1-GlyRs (25, 123, 139, 212, 227). However,

one study that examined the equilibrium gating of recom-

binant ␣1-GlyRs identified two further burst-length com-

ponents, namely, a particularly short-lived state that was

prominent at low glycine concentrations and a particu-

larly long-lived state that was seen only at the highesttested concentrations (25). The authors interpreted these

results by proposing that the shortest to longest lived

states correspond to receptors occupied by one to fi ve

glycine molecules, respectively. These findings were rec-

onciled into an MWC-like model where any one of fi ve

possible liganded states can lead to channel opening. An

attractive feature of this model is that ␣1-GlyRs do indeed

possess fi ve glycine-binding sites.

However, this model is controversial because it pre-

dicts that mono-liganded openings should add an instan-

taneous linear component to the onset of the glycine-

stimulated response, and experiments involving the rapid

application of glycine to both native and recombinantGlyRs have revealed the distinct absence of such a com-

ponent (128, 139, 218, 227). The shape of the glycine

activation response was consistent with a minimum of

two bound glycines being required to activate the native

GlyR (218) or a minimum of two or three bound glycines

for activation of the recombinant ␣1-GlyRs (128, 139,

227). Thus the GlyR kinetic models generated by analysis

of stationary and nonstationary receptor kinetics cannot

be resolved at present. Because such models are crucial

for understanding and predicting receptor behavior, it is

hoped that both stationary and nonstationary kinetic anal-

yses will be included in the development and functionaltesting of future models.

In contrast to the ␣1-GlyR, the ␣2-GlyR can be mod-

eled as possessing only a single open state (246). It also

has an extraordinarily slow rate of receptor activation

that requires at least two simultaneously bound glycine

molecules (246). Mangin et al. (246) successfully modeled

these properties by proposing that the single open state

was linked to the fully liganded closed state.

Transitions to and from desensitized states have also

been modeled in ␣1- (128, 218, 246) and ␣2-GlyRs (246).

These models depict these states as being accessed from

the singly or doubly liganded closed states. Some kinetic

studies have ignored the effects of desensitization be-

cause it generally occurs at a relatively slow rate.

C. Agonist Binding

1. Agonist af fi nity and ef fi cacy

Native and recombinant GlyRs are activated by

amino acid agonists with the following rank order of

potency: glycine Ͼ ␤-alanine Ͼ taurine Ͼ GABA. In ␣1-

GlyRs expressed in HEK293 cells, glycine, ␤-alanine, and

taurine all behave as full agonists, with glycine exhibiting

an EC50 value of 20 –50 M (46, 237). However, when the

same ␣1-GlyRs are expressed in Xenopus oocytes, the

EC50 values of all agonists are increased by about an

order of magnitude, and the peak magnitudes of saturat-

ing ␤-alanine-, taurine- and GABA-gated currents are re-

duced relative to those activated by glycine (214). Thesedifferences, shown in diagrammatic form in Figure 4,

imply variations in GlyR conformation between the two

expression systems. A recent detailed study of Xenopus

oocyte-expressed ␣1- and ␣2-GlyRs showed that the gly-

cine, taurine, and GABA sensitivity varied in parallel from

cell to cell over a surprisingly large (10-fold) range (90). In

addition, the relative peak magnitudes of taurine- and

GABA-gated currents varied according to their EC50 val-

ues. The origin of this variability is not known, but such a

degree of variability has not been reported in GlyRs ex-

pressed in HEK293 cells.

The information available to date suggests that ␣2-,

␣3-, and ␣4-GlyRs exhibit similar agonist sensitivities tothe ␣1-GlyR (90, 130, 157, 201, 202, 246). The incorpora-

FIG. 4. Typical agonist sensitivity profiles of human ␣1 GlyRsrecombinantly expressed in HEK293 cells (top) and Xenopus oocytes(bottom).





tion of ␤-subunits has little effect on receptor sensitivity

to glycine (154, 298, 268), although its effect on the EC50

values for other agonists has not been determined to date.

A combination of rapid agonist application tech-

niques and equilibrium single-channel kinetic analysis

was used by Lewis et al. (227) to estimate the agonist

af finities and ef ficacies of glycine, ␤-alanine, and taurineon ␣1-GlyRs expressed in HEK293 cells. The respective

K A values were estimated as follows: glycine, ϳ160 M;

␤-alanine, ϳ360 M; and taurine, ϳ460 M. These differ-

ences were found to be entirely due to variations in the

agonist dissociation rates. The E values were predicted to

be 16, 8.4, and 3.4 for glycine, ␤-alanine, and taurine,

respectively (227). The differences among these values

were caused by variations in the channel opening rate,

with the channel closing rate remaining constant for all

three agonists. Earlier studies on homomeric ␣1-GlyRs

(128) and heteromeric ␣1␤-GlyRs (218) estimated compa-

rable E and K A values for glycine. In contrast, the homo-meric ␣2-GlyR was estimated to have a glycine E value of

at least 250 and a K A value near 40,000 M (246).

2. Agonist binding domains

As discussed in detail in section IIIC , the ligand-bind-

ing pocket is formed as a cleft between adjacent subunits.

Three loops (domains A, B, and C) form the “ principal”ligand-binding surface on the ϩ side of the interface (Fig.

3 A) and three ␤-strands (domains D, E, and F) from the

adjacent subunit comprise the “complementary” (or Ϫ)

face. The residues of AChBP that contribute to thesedomains are indicated in Figure 2. The human GlyR ␣1-

and ␤-subunit residues that align with the AChBP ligand-

binding site residues are also shown.

Kuhse et al. (202) observed that the GlyR ␣2*-splice

variant subunit had a glycine EC50 that was ϳ40 times

higher than those of the ␣2- or ␣1-subunits. Glycine sen-

sitivity was restored by incorporating the E167G mutation

into the ␣2*-subunit, thus reversing the only noncon-

served amino acid between the ␣2*- and ␣2-subunits. This

residue aligns with G160 in agonist binding domain B of

the GlyR ␣1-subunit (Fig. 2). It was subsequently shown

that the double mutant F159Y, Y161F ␣1-GlyR caused a modest (12-fold) increase in glycine sensitivity, but sur-

prisingly large (121- and 45-fold) increases in the sensitiv-

ities to ␤-alanine and taurine (338). Although EC50

changes alone do not provide evidence for binding inter-

actions, domain B was considered likely to bind glycine

since 1) the mutation also dramatically affected GlyR

sensitivity to the competitive antagonist strychnine (see

below), and 2) this domain had already been identified as

an nAChR binding site (reviewed in Ref. 19). The domain

has since been implicated in agonist binding in the GABA A and 5-HT3 receptors (14, 354).

The agonist-binding role of the GlyR ␣1-subunit C

domain has been investigated by the Schofield group (309,

381, 382). The GlyR is unusual among LGICs in that its C

domain is contained within a second extracellular disul-

fide loop. It was shown that mutations to the even-num-

bered residues, L200, Y202, and T204, profoundly affect

receptor sensitivity to glycine or strychnine, or both (309,381, 382). Of particular note, the conservative Y202F mu-

tation caused a drastic increase (480-fold) in the glycine

EC50, while having little effect on strychnine sensitivity

(309). Due to the differential sensitivity of specific resi-

dues to glycine and strychnine and the fact that this

domain had been implicated in acetylcholine binding to

the nAChR (reviewed in Ref. 19), binding domain C is

likely to be involved in glycine binding. However, most

mutations to this domain also affected receptor gating, as

evidenced by their conversion of taurine from a full into a

partial agonist (309).

Residues I93 and N102 in domain A were identified asagonist-binding elements on the grounds that conserva-

tive mutations affected agonist EC50 values only, without

affecting strychnine sensitivity or the agonist ef ficacies of

␤-alanine or taurine (151, 379). The R97G mutation, which

induced spontaneous gating in ␣1-GlyRs (32), may also

have exerted a direct or allosteric effect on this site.

The possible agonist-binding roles of the GlyR ␣-sub-

unit complementary (D, E, and F) domains have not yet

been investigated. This constitutes a significant gap in our

understanding of glycine binding mechanisms, as it is

currently unclear whether bound glycine molecules are

coordinated by adjacent subunits.

The possible agonist-binding role of the ␤-subunithas also received scant attention. A study on Xenopus

oocyte-expressed GlyRs found that the incorporation of

␤-subunits reduced the Hill coef ficient for glycine activa-

tion from 4.2 in ␣1-homomers to 2.4 in ␣1␤-heteromers

(46). This implied that ␤-subunits did not contain glycine

binding sites. However, not all studies have observed a

change in Hill coef ficient upon ␤-subunit incorporation

(e.g., Ref. 154). Interestingly, a single-channel study inves-

tigating the effect of the ␤-subunit startle disease muta-

tion G229D found that it disrupted the agonist binding

af finity (314). Because G229 lies in loop C (Fig. 2), this

effect was most likely mediated by either a direct or allosteric disruption of a ␤-subunit glycine binding site.

Clearly, further experiments are required to clarify the

agonist-binding role of the ␤-subunit.

3. Physical basis of agonist binding

There is abundant evidence that the ACh-nAChR

binding reaction is mediated mainly by noncovalent “cat-

ion- ” electrostatic interactions (430). In this system, the

aromatic side chains of phenylalanine, tyrosine, or tryp-

tophan contribute a negatively charged surface while





the cation is provided by the agonist. In the ␣1-nAChR

subunit, the ACh quaternary nitrogen interacts directly

with Y149 by this mechanism (434). The corresponding

GlyR ␣1-subunit residue Y161 is conserved in all GlyR

subunits. By analogy with the nAChR, Y161 could con-

ceivably form a cation interaction with the glycine

amine nitrogen.Glycinergic agonists have been subjected to a num-

ber of structure-activity investigations, with the most no-

table being that of Schmieden and Betz (336). This study

tested the agonist and antagonist potencies of a range of

␣- and ␤-amino acids. Agonist activity was found exclu-

sively to be a property of those molecules where the

amino and acidic moieties existed in a cis-conformation

(Fig. 5). One molecule (nipecotic acid) that was locked

into a trans-conformation exhibited only antagonist ac-

tivity. However, ␤-amino acids (e.g., taurine) that ran-

domly flicker between both conformations displayed both

agonist and antagonist activity (Fig. 5). This model pre-

dicts a specific antagonist recognition site in a common

ligand binding pocket that is accessible only by trans-

isomers (336). By binding in the trans-conformation,

␤-amino acids may either stabilize the closed state or

sterically prevent glycine from binding in the pocket, or

both. This model also predicts that the partial agonist

activity of taurine results from a fraction of molecules

binding as antagonists and another fraction binding as

agonists at the same receptor. Although attempts to iden-

tify a putative antagonist contact site have proven incon-

clusive to date (151, 339), this interesting model is cer-

tainly worthy of further consideration.

D. Structural Changes Accompanying Activation

1. The ligand-binding domain

Functional data suggest that nAChR activation is me-

diated by global intersubunit movements (74). A recent

structural analysis of the nAChR by Unwin et al. (377) has

provided more direct support for such a model. This

group interpreted low-resolution electron diffraction im-

ages of Torpedo nAChRs obtained in both the closed and

open states using the AChBP crystal structure as a tem-

plate. In modeling the structural changes, they divided the

␣-subunit into inner and outer parts and considered each

separately. The inner part (which includes all residues up

to the conserved cysteine loop) is so-called because it

faces the channel vestibule and contains most of the

intersubunit contact points plus agonist-binding domain

A. The outer part (which comprises ␤-sheets 7–10) in-

cludes the agonist-binding domains B and C. Upon agonist

binding, the outer part was found to undergo an upwardstilt around an axis parallel with the membrane plane.

Simultaneously, the inner part rotated ϳ15° in a clock-

wise direction (when viewed from the synapse) around an

axis perpendicular to the membrane plane. Being essen-

tially a combination of rigid body movements, the largest

residue displacements (up to 2 Å ) occurred at the subunit

interfaces. The rotation of the inner sheets is viewed as

the crucial event in transmitting agonist-binding informa-

tion to the channel gate (377). The inner sheets contain

two loops, the conserved cysteine loop and loop linking

the first and second ␤-sheets (referred to hereafter as loop

2), that are ideally located to interact directly with theTM2-TM3 domain (52, 267). Interactions between these

loops and the TM2-TM3 domain appear crucial to the

GlyR activation process, and the extensive body of evi-

dence implicating these regions in receptor gating will

now be considered.

2. The TM2-TM3 domain

The structural changes initiated in the ligand-binding

domain are transmitted to the membrane-spanning do-

mains, where they culminate in a change in conformation

of TM2. As stated above, one such contact point is the

“TM2-TM3 domain” which extends from R271 to D284 inthe GlyR ␣1-subunit. Although originally thought to com-

prise an extended extramembranous loop, the Miyazawa

TM structure now reveals this domain to comprise the

extramembranous portions of the TM2 and TM3 ␣-helices

plus a short connecting loop (267).

A signal transduction role for single residues in this

domain was first suggested by studies on the GABA CR

(204), the GlyR (305), and the nAChR (56). A systematic

study subsequently proposed a structural role for the

entire ␣1-GlyR TM2-TM3 domain in the gating process

(237). This conclusion was based on the observation that

FIG. 5. Structural comparison of GlyR agonists and antagonists.Ligands (glycine and L-alanine) that display only agonist activity are

shown on the left. Nipecotic acid, which displays only antagonist activ-ity, is shown on the right. Taurine, which displays both agonist andantagonist activity, flickers randomly between the two indicated confor-

mations. In the cis-conformation, the distance between amino andacidic groups is similar to that found in glycine. In the trans-conforma-tion, the distance is similar to that seen for nipecotic acid.





insight into how these interactions might occur. The

structure suggests the existence of hydrophobic bonds

between the 9Ј and 10Ј residues of adjacent subunits.

These bonds appear to “balance” the L9Ј residues into a

fi vefold radially symmetrical arrangement that holds the

channel closed. It is likely that agonist-induced conforma-

tional changes asymmetrically disrupt some of thesebonds, leading to a collapse of symmetry and a simulta-

neous conversion of all TM2 domains to the activated

state. Mutations to one or more L9Ј residues may achieve

a similar effect.

In summary, the main features of agonist-induced

TM2 movements are that 1) its midpoint acts as a swivel,

and 2) this swivel point mediates interactions between

adjacent subunits. Agonist-induced backbone rearrange-

ments at this position may thereby lead to a concerted

conformational change at either a centrally located or an

intracellularly located gate (376, 404).

Functional evidence suggests that TMs 1, 3, and 4may also be involved in LGIC gating. Single-channel ki-

netic analysis of mutations incorporated into each of

these domains suggests that some residues may have

specific roles in gating the nAChR (47, 89, 104, 388). SCAM

analysis has also provided evidence for state-dependent

structural rearrangements of TM1 and TM3 in the nAChR

and GABA A R, respectively (6, 400 – 402, 432). However,

insuf ficient information is available to date on any LGIC

member to form a coherent picture of how these domains

contribute to activation. The recently elucidated nAChR

TM domain structure (267) now permits the design of

more specific experiments to investigate these mecha-

nisms.

5. The ␤-subunit

The role of the ␤-subunit in ␣1␤-GlyR gating was

recently investigated by incorporating mutations into cor-

responding positions in ␣1- and ␤-subunits and comparing

their effects on receptor function (344). Although cys-

teine-substitution mutations to residues in the NH2-termi-

nal half of the ␣1-subunit TM2-TM3 loop dramatically

impaired gating ef ficacy (234), the same mutations ex-

erted little effect when incorporated into corresponding

positions of the ␤-subunit (344). Furthermore, although␣1-subunit TM2-TM3 loop cysteines were modified by

cysteine-specific reagents (234), the corresponding ␤-sub-

unit cysteines showed no evidence of reactivity (344).

These observations suggest structural or functional dif-

ferences between ␣1- and ␤-subunits. However, the incor-

poration of the L9ЈT mutation into the ␤-subunit dramat-

ically increased the glycine sensitivity (344), suggesting

an allosteric modulatory effect on the ␣1-subunit. Thus

␤-subunit conformational changes do contribute to the

activation of the GlyR, although their involvement in this

process is significantly different to that of the ␣1-subunit.

VI. GLYCINE RECEPTOR MODULATION

A. Phosphorylation

Phosphorylation can cause long-term changes in the

functional properties of ion channels, and an abundance

of evidence implicates such mechanisms in various forms

of synaptic plasticity (177). Intracellular signaling path-

ways determine the phosphorylation state of proteins by

coordinating the activities of protein kinases, which in-

duce phosphorylation, and phosphatases, which reverse

it. All of the functional phosphorylation sites on LGICs

have been mapped to the major intracellular loop (360).

As discussed above, LGIC subunits exhibit a low degree

of sequence homology in this region, and this underlies

the subunit-specific distribution of phosphorylation con-

sensus sites (Fig. 2). It has recently become apparent that

clustering and cytoskeletal anchoring proteins can influ-

ence the proximity of kinases and phosphatases to theLGIC intracellular domains (226, 360). Thus the ability of

an LGIC to be phosphorylated depends not only on its

subunit composition but also its proximity to the appro-

priate enzymes. In addition, kinases and phosphatases

may have indirect effects on the GlyR by controlling the

phosphorylation state of modulatory proteins. This diver-

sity could lead to tissue-specific differences in the pro-

pensity of a given GlyR isoform to be phosphorylated.

1. Protein kinase A

Contrasting effects of cAMP-dependent phosphoryla-

tion on GlyR current magnitudes have been reported inneurons from various parts of the brain (219). Although

the differences may be related to the phosphorylation of

GlyR modulatory proteins (or possibly to tissue-specific

or nonspecific effects of pharmacological probes), bio-

chemical experiments strongly suggest that spinal cord

GlyR ␣-subunits themselves are directly phosphorylated

in vitro (378). However, most GlyR ␣-subunit isoforms

do not contain protein kinase A (PKA) phosphorylation

consensus sequences. The exception to this is the ␣1ins

splice variant which contains an eight-amino acid insert

(. . . SPMLNLFQ. . . ) in the large intracellular domain, and

the first residue of this insert may serve as a phosphory-lation site (244). As shown in Figure 2, the ␤-subunit also

contains a PKA consensus site at a different position

(T363) in the TM3-TM4 domain (137). However, it is yet to

be determined whether the ␣1ins or the ␤-subunit sites are

directly phosphorylated and, if so, whether phosphoryla-

tion induces changes in receptor function. As noted by

Legendre (219), it is possible that the contradictory ef-

fects of cAMP-dependent phosphorylation may be ex-

plained by the differential expression of ␣1ins- and ␤-sub-

units throughout the central nervous system (245).

Whether this is true, and whether cAMP-dependent phos-





phorylation has a physiological role at glycinergic syn-

apses, are important questions for future research.

2. Protein kinase C

Physiologically, protein kinase C (PKC) is activated

by increases in intracellular calcium or diacylglycerol.The GlyR ␣1-subunit contains a PKC phosphorylation

consensus sequence at S391 in the TM4 domain (323).

Consistent with this, the spinal cord GlyR ␣-subunit was

shown to be phosphorylated by PKC in an in vitro assay

(378). Support for a functional role for S391 is provided by

the observation that the homologous residue in the

GABA A R ␤-subunit is phosphorylated by PKA and PKC

(274). Surprisingly, however, the corresponding residue in

the Torpedo nAChR appears inaccessible to the protein

surface (267). The GlyR ␤-subunit also contains a putative

PKC phosphorylation consensus site at position 389 in the

TM3-TM4 domain (137), although its functional status is yet to be confirmed. Pharmacological manipulations

aimed at stimulating or inhibiting PKC have revealed con-

trasting effects on glycine-activated currents in a variety

of neuron types (219). As discussed above, these differ-

ential effects may be due to a multitude of causes, includ-

ing, in some cases, nonspecific effects of pharmacological

agents (e.g., Ref. 281). Our current understanding of PKC-

dependent phosphorylation is further complicated by the

fact that differences have also been observed in suppos-

edly identical recombinant GlyRs. For example, PKC was

found to potentiate glycine currents in Xenopus oocyte-

expressed ␣1- and ␣2-homomeric GlyRs (281). In appar-ent contrast, PKC activators did not affect current mag-

nitude in ␣1-GlyRs expressed in either Xenopus oocytes

(253) or HEK293 cells (128). However, the later study

reported that PKC activation accelerated the onset of and

slowed the recovery from desensitization (128). A defini-

tive understanding of PKC-dependent phosphorylation

processes in the GlyR will require functional assays in-

volving site-directed mutagenesis of putative phosphory-

lation sites and biochemical assays to directly determine

the receptor phosphorylation state.

Interestingly, PKC activation has been shown to in-

crease the potentiating effects of ethanol in recombinant␣1-GlyRs (253). Ethanol was shown to potentiate, inhibit,

or have no effect on glycine-activated responses in 35, 45,

and 20%, respectively, of a large sample of ventral teg-

mental area (VTA) neurons (422, 423). In the population

of VTA neurons where ethanol induced inhibition, PKC

activators seem to compete with ethanol for a common

inhibitory site (364). A pharmacological study on those

VTA neurons where ethanol induced potentiation, activa-

tion of the PKC epsilon isoform was found to increase

potentiation magnitude (173). Together, these findings

raise the possibility of a specific allosteric linkage be-

tween the phosphorylation site and the alcohol binding

site.

3. Protein tyrosine kinase

Lavendustin A, a protein tyrosine kinase (PTK) inhib-

itor, reduced glycine currents in hippocampal and spinalneurons, whereas intracellular application of c-Src, an

endogenous tyrosine kinase, increased glycine current

magnitudes (57). The current increases, which were me-

diated by a leftward shift in the glycine EC50, were ac-

companied by an enhanced desensitization rate (57). Sim-

ilar effects of c-Src were observed on recombinant ␣1␤-

GlyRs expressed in HEK293 cells, but not in recombinant

␣1-GlyRs expressed in the same cells. Mutation of a pu-

tative tyrosine phosphorylation site (Y413F) in the large

intracellular loop of the ␤-subunit (137) abolished the

effects of several tyrosine phosphorylation modulators,

suggesting this site is functionally phosphorylated (57).

Although these experiments provide strong circumstan-

tial evidence for ␤-subunit phosphorylation, biochemical

confirmation is required to eliminate the possibility of

allosteric effects induced by the Y413F mutation.

B. Modulators of Possible Physiological Relevance

1. Zinc

A ) A PUTATIVE PHYSIOLOGICAL ROLE. Zinc is concentrated

into round clear presynaptic vesicles in the central ner-

vous system and is released into the synaptic cleft by

nerve terminal stimulation (20, 120, 164). During synapticstimulation, zinc is thought to reach a peak external con-

centration of Ͼ100 M (20, 120, 385). At such concentra-

tions, zinc is able to modulate a wide variety of pre- and

postsynaptic ion channels (349). Low (0.01–10 M) con-

centrations of zinc potentiate glycinergic currents by in-

creasing the apparent glycine af finity without changing

the saturating current magnitude, whereas higher concen-

trations (Ͼ10 M) inhibit the current by reducing the

apparent glycine af finity (213). This pattern of zinc action

is seen in native receptors (42, 63, 152, 359, 365) as well as

in recombinant ␣1-, ␣2-, and ␣1␤-GlyRs (213). In addition

to increasing the magnitude of glycinergic IPSCs, lowconcentrations of zinc have also been shown to prolong

their duration and frequency (211, 359), with both effects

presumably being due to the increased glycine sensitivity.

The role of zinc has been most thoroughly investi-

gated at the mossy fiber glutaminergic synapse in the

hippocampus (120). However, some suggestions have re-

cently emerged that zinc may also have a physiological

role at glycinergic synapses. First, an ultrastructural study

has found evidence for zinc and glycine colocalization in

individual presynaptic terminals in the spinal cord (39).

Second, at glycinergic synapses of the intact zebra fish





hindbrain, zinc chelators decreased the amplitude, dura-

tion, and frequency of glycinergic IPSCs, whereas zinc

application had the opposite effect (359). However, it

remains to be established whether zinc is coreleased with

glycine at concentrations high enough to modulate GlyR

function.

It is important to note that even strongly inhibitingzinc concentrations (Ն30 M) causes an initial transient

potentiation followed by the slowly developing inhibition

(235). The duration of this transient potentiation, which is

of the order of 1 s (203, 235), easily exceeds that of a

typical glycinergic IPSC. However, zinc inhibition stabi-

lizes much more rapidly in the absence of agonist (235) so

that if an inhibiting concentration of zinc reaches the

GlyR before glycine does, then only inhibition is seen

(211, 235). Together, these observations mean that high

zinc concentrations may have opposite effects on glycin-

ergic IPSC magnitude depending on whether the zinc

reaches the receptor before or after glycine. This shouldbe considered in models of zinc action on glycinergic

synapses.

No other metal ion has convincingly been shown to

mimic the biphasic action of zinc. However, the potenti-

ating site is recognized by several metal ions with the

potency sequence: zinc Ͼ lanthanide Ͼ lead Ͼ cobalt (96,

203), whereas the inhibitory site exhibits the potency

sequence: zinc Ͼ copper Ͼ nickel (96, 203).

B) MOLECULAR MECHANISM OF ACTION. In most proteins,

zinc ions are coordinated by nitrogen, sulfur, or oxygen

atoms found in the side chains of histidine, cysteine,

aspartic acid, and glutamatic acid residues (131). Zinc

binding sites are usually comprised of two to four resi-dues, and the af finity of a site depends on the number of

residues, their relative positions, and the local electro-

static environment (21, 317). Several lines of evidence

suggest that the GlyR zinc potentiating and inhibitory

binding sites are physically discrete.

C) INHIBITION. In contrast to the rapid onset of poten-

tiation, zinc inhibition is slow to develop (235). However,

as noted above, this inhibition develops much more rap-

idly in the absence of agonist (235). This result suggests

that glycine-induced activation is accompanied by a struc-

tural change in this location and that zinc acts by stabi-

lizing the closed conformation. A single-channel studyfound high (50 M) zinc concentrations reduced ␣1-GlyR

open probability by reducing mean channel open time and

the relative abundance of long channel bursts (212). It

concluded that zinc increases the rate at which the chan-

nel exits from the open state, supporting the view that

zinc stabilizes the closed state.

Zinc inhibition of the recombinant ␣1-GlyR was

found to be selectively abolished by either reducing the

pH or by pretreatment with diethylpyrocarbonate, a his-

tidine-specific modifying agent (158). Because both treat-

ments effectively reduce the ability of zinc to bind with

histidine imidazole rings, histidines were implicated in the

complexation of zinc at its inhibitory site. Mutations to

either H107 or H109 were subsequently shown to abolish

zinc inhibition, strengthening the case that these residues

formed an inhibitory binding site (158). Histidine ␣-car-

bonyl oxygen atoms need to be within 13 Å of each other

to permit their imidazole rings to coordinate a zinc ion(21). Because the histidines are separated by only one

residue, it is certainly feasible that the zinc ions could be

coordinated within individual ␣-subunits. However, the

␣-carbonyl oxygens of the homologous residues in adja-

cent AChBP subunits are separated by 7.7 Å (52). With the

assumption that this structure is reasonably well con-

served in the GlyR, it is plausible that zinc ions could be

coordinated by adjacent ␣-subunits. This possibility was

tested by coexpressing ␣1-subunits containing the H107A

mutation with ␣1-subunits containing the H109A mutation

(276). Although sensitivity to zinc inhibition is markedly

reduced when either mutation is individually incorpo-rated into all fi ve subunits, the GlyRs formed by the

coexpression of H107A mutant subunits with H109A mu-

tant subunits exhibited an inhibitory zinc sensitivity sim-

ilar to that of the wild-type ␣1-homomeric GlyR. This

constitutes strong evidence that inhibitory zinc is coordi-

nated at the interface between adjacent ␣1-subunits, but

does not rule out the possibility that zinc may also be

coordinated within ␣1-subunits. No evidence was found

for ␤-subunit involvement in the coordination of inhibi-

tory zinc, indicating that a maximum of two zinc-binding

sites per heteromeric receptor is suf ficient for maximal

zinc inhibition (276). This region of the ␣1-subunit aligns

poorly with AChBP due to the existence of three addi-tional ␣1-subunit residues. Homology models were con-

structed using several alignments, and only one of these

produced a plausible zinc binding site (276). The success-

ful alignment is depicted in Figure 2, and the modeled

structure of this site is shown in Figure 3 B.

D) POTENTIATION. Low (5 M) zinc concentrations in-

crease the open probability of the ␣1-GlyR by increasing

both the opening frequency and the mean burst duration

(212). The authors concluded that the channel opening

and closing rates were not significantly affected and that

zinc acted primarily by slowing the rate of glycine disso-

ciation from the binding site. This suggests an allostericeffect of zinc that improves the fit of glycine to its site. In

contrast, zinc had a dual effect on taurine-gated currents.

It not only slowed the rate of taurine dissociation from its

binding site, but increased the rate at which bound tau-

rine could activate the channel (212). Another group

showed that mutations to various residues in the TM1-

TM2 and TM2-TM3 domains abolished zinc potentiation

of glycine currents, while leaving zinc potentiation of

taurine currents intact (235). These results suggest that

the allosteric linkage between the zinc potentiating site

and the glycine binding site or transduction pathway was





selectively disrupted. The results can be reconciled with

those of Laube et al. (212) by proposing that the muta-

tions selectively disrupted the ability of zinc to enhance

the glycine af finity.

Analysis of chimeric constructs of ␣1- and ␤-subunits

implicated D80 as a specific determinant of zinc potenti-

ation (213). Indeed, mutations (D80A, D80G) to this resi-due disrupted zinc potentiation of glycine currents (212,

235), whereas mutations to neighboring aspartic acid res-

idues (D81A, D84A) had no effect (212). However, be-

cause D80A did not abolish zinc potentiation of taurine-

gated currents (235), its putative role as a zinc binding site

must be queried. The potentiating effect of zinc was also

abolished by the H109A mutation (158) as well as several

mutations in the TM1-TM2 and TM2-TM3 linker domains

(235). Unfortunately, this widespread distribution of site-

directed mutations that abolish zinc potentiation does not

bode well for the use of this approach in identifying the

GlyR zinc potentiating site. The lack of zinc voltage dependence and rapid reversibility of the potentiating effect

(212, 235) indicate that the potentiating binding site re-

sides in an extracellular domain.

2. Calcium

Calcium current influx through glutamate-activated

channels causes a rapid (Ͻ100 ms) and transient eleva-

tion of glycine current magnitude (124, 411, 412) that may

have an important physiological role in modulating the

gain of glycinergic transmission. Based on a pharmaco-

logical analysis on GlyRs expressed on rat spinal sensory

neurons, Xu and colleagues (411, 412) proposed that theeffect was mediated by calcium activation of calmodulin-

dependent protein kinase II and calcineurin. However,

Fucile et al. (124), who examined a similar effect in both

cultured spinal neurons and recombinant ␣1-GlyRs, found

it was resistant to a variety of manipulations designed to

disrupt phosphorylation, dephosphorylation, and G pro-

tein-dependent processes. These differences may relate to

the different origins of the neurons studied and could be

indicative of multiple mechanisms contributing to this

effect. Another recent study, conducted on VTA neurons,

found the calcium-dependent potentiation to be antago-

nized by ethanol (438).In the effect seen by Fucile et al. (124), single-channel

analysis suggested the calcium-dependent factor exerted

complex effects on both the glycine binding af finity and

the gating rate (124). Because the calcium-induced in-

crease did not reverse in inside-out patches, it was con-

sidered likely to be mediated by the calcium-induced

removal of a soluble cytoplasmic intermediate from the

receptor internal surface. Although the identity of this

intermediate remains elusive, it appears to be endog-

enously expressed in HEK293 cells as well as in spinal

neurons (124).

3. pH

Transient increases in extracellular pH occur in re-

sponse to the activation of anionic LGIC receptors (65).

The mechanism is most likely related to the high HCO3

Ϫ

permeability of anionic LGICs. When the pore opens it is

likely that HCO3

Ϫ exit the cell, causing intracellular acid-

ification and extracellular alkalization (65). In recombi-

nant ␣1- and ␣1␤-GlyRs, the glycine EC50 is significantly

increased as the pH is lowered from 7.5 to 6.0 (64). This

effect appears to be mediated by a specific interaction

with the GlyR extracellular domain as it is abolished by

the ␣1-subunit mutations H109A, T112A, and T112F, but is

not affected by other mutations to T112 or by mutations

to neighboring negatively charged residues (64). Mutation

to the ␤-subunit residue that corresponds to T112 (i.e.,

T135A) was also found to reduce proton sensitivity (64).

Thus pH sensitivity appears to be specific to the receptor

region that houses the zinc inhibitory binding site. How-

ever, as threonines are not ionizable, it is unlikely thatthey directly form the proton acceptor site.

4. Neurosteroids

Neurosteroids are hormones that are synthesized in

central nervous system glia and neurons from cholesterol

or blood-borne steroidal precursors (24, 357, 372). Al-

though neuroactive steroids produced in the peripheral

steroidogenic glands can easily access the brain, it is

thought that steroids produced in the central nervous

system have important paracrine roles (207). Although

the complex behavioral effects of neurosteroids have

been attributed primarily to GABA A and NMDA receptors(24, 103, 207, 357), potent neurosteroid actions have been

observed on native and recombinant GlyRs. Antagonistic

effects of progesterone, its precursor pregnenalone

(PREG), and pregnenolone sulfate (PREGS) were first

shown on glycine currents in cultured spinal neurons

(407, 408). Another early report showed that glycine sen-

sitivity in the rat optic nerve was increased by several

corticosteroids at concentrations of 1–10 M (299). On

the other hand, both ␣1- and ␣1␤-GlyRs were found to be

insensitive to the action of 5␣-pregnen-3␣-ol-20-one (295).

Using a more systematic approach, Maksay et al. (243)

examined a battery of neurosteroids on recombinant ␣1-,␣2-, ␣4-, ␣1␤-, and ␣2␤-GlyRs. Both PREGS and dehydro-

epiandrosterone sulfate (DHEAS) inhibited all of these

receptors with inhibitory constant ( K i) values of 2–20 M,

with the compounds showing only a modest degree of

subunit specificity. Of particular note, DHEAS inhibited

the ␣2-GlyR with an IC50 of 4.2 M, whereas its IC50 at the

␣1␤-GlyR was 21.2 M. PREG caused an ϳ25% increase in

the ␣1-GlyR current with an EC50 of 1.4 M, but had no

effect on ␣1␤- or ␣2-GlyRs (243). On the other hand,

progesterone inhibited the ␣2-GlyR current by 23% with

an IC50 of 20 M, while exerting no effect on the ␣1- and





␣1␤-GlyRs (243). Given the ␣2 3 ␣1␤ subunit switch that

occurs after birth in the rat, the differential effects of

DHEAS and progesterone on the ␣2- and ␣1␤-GlyRs may

have physiological relevance for neuronal development.

Although the molecular determinants of neurosteroid

action at the GlyR have not yet been determined, some

progress has been made on the GABA A R. On the basis of a chimeric study on alphalaxone-sensitive and -insensitive

GABA A R subunits, a necessary determinant of neuroste-

roid action was isolated to the NH2-terminal half of TM2

(318). Consistent with this, the V2’S mutation in the

GABA A R ␣1-subunit reduced the potency of PREGS block

by 30-fold (11). Particularly since the GlyR 2Ј residue is

known to affect the potencies of other GlyR modulators

(see below), it is a promising candidate as a specific

determinant of neurosteroid action in the GlyR.

Finally, it has long been known that that the synthetic

steroid RU 5135 displaces [3H]strychnine binding with a

remarkably high (5 nM) af finity (48). Because the mech-anism of action of this ligand has never been investigated,

it is not known whether it shares a similar mode of action

to the endogenous neurosteroids.

5. G protein ␤␥ -subunits

The irreversible activation of G proteins by nonhy-

drolyzable GTP analogs has recently been shown to exert

potent effects on the GlyR. These reagents cause both a

leftward shift in the glycine EC50 of recombinant ␣1-

GlyRs and a prolongation of glycinergic synaptic currents

in cultured spinal neurons (425). These effects are most

likely mediated by a direct interaction of G protein ␤␥ -subunits with the GlyR ␣1-subunit because 1) ␤␥ -subunits

coimmunoprecipitate with the GlyR ␣1-subunit, and 2)

addition of ␤␥ -subunits to the intracellular membrane

surface reversibly increases GlyR channel activity (425).

These results suggest that G protein-coupled receptor

activation may be important in vivo for regulating the gain

of glycinergic synaptic transmission.

C. Molecular Pharmacology

1. Strychnine and analogs

The plant alkaloid strychnine (Fig. 6) is a highly

selective and extremely potent competitive antagonist of

glycine, ␤-alanine, and taurine with a dissociation con-

stant of 5–10 nM (81, 427, 428). Strychnine sensitivity is

currently the most definitive means of discriminating gly-

cinergic from GABAergic synaptic currents. Because of

its high af finity and specificity, [3H]strychnine has been

widely used in radioligand displacement studies to inves-

tigate the potencies and allosteric actions of other GlyR

ligands (241). Strychnine has been subjected to several

structure-activity investigations (162, 239, 249). However,

no analog has ever been shown to possess a higher ap-

parent af finity than strychnine. Similarly, no strychnine-

like ligands have yet been shown to exert agonist or

allosterically enhancing properties. Surprisingly, how-

ever, strychnine has been shown to behave as an agonist

in modified ␣7-homomeric nAChRs (286).

Early evidence from GlyR protein modification experiments indicated that the strychnine and glycine bind-

ing sites were mutually interactive but not identical (249).

This interpretation has also been reached as a result of

site-directed mutagenesis experiments. The first of these

experiments stemmed from the observation that the ␣2*-

splice variant subunit had a strychnine IC50 that was ϳ560

times higher than those of the ␣2- or ␣1-subunits (202).

Strychnine sensitivity was restored by incorporating the

E167G mutation into the ␣2*-subunit, thus reversing the

only nonconserved amino acid between the ␣2*- and ␣2-

subunits (202). This residue aligns with G160 in the GlyR

␣1-subunit. As expected, strychnine insensitivity was con-ferred to the ␣1-subunit by the reverse G160E mutation

(381). Mutation to the adjacent ␣1-subunit residue Y161A

also abolished strychnine sensitivity (381), although the

more conservative mutation Y161F did not (338). Photoaf-

finity labeling experiments localized another crucial

strychnine binding element to either Y197 or Y202 of the

␣-subunit (322). Subsequently, Vandenberg and col-

leagues (309, 381, 382) identified K200 and Y202 as strych-

nine binding sites on the basis of functional analysis of

␣1-GlyRs incorporating site-directed mutations at these

positions. Because glycine also binds to common or ad-

jacent residues in both of these strychnine-binding do-

mains (see above), it is reasonable to conclude thatstrychnine and glycine share overlapping but nonidentical

binding sites in principal ligand binding domains B and C.

This, of course, provides a structural basis for the com-

petitive antagonist behavior of strychnine (382).

Substances that act purely as competitive antago-

nists are comparatively rare. Generally, it would be ex-

pected that any substance that binds to a receptor would

also bias the conformational equilibrium towards a par-

ticular state. However, the possible allosteric effects of

strychnine have yet to be considered. One way of doing so

would be to determine the effects of low strychnine con-

centrations on modified GlyRs that spontaneously gate inthe absence of agonist.

2. Picrotoxin and analogs

Derived from plants of the moonseed family, the

convulsant alkaloid picrotoxin is also widely used to dis-

criminate GABAergic from glycinergic currents (348). Pi-

crotoxin (PTX) strongly inhibits GABA A Rs at 1–10 M

concentrations, whereas GlyRs in vivo are considerably

less sensitive. PTX comprises an equimolar mixture of

picrotoxinin and picrotin. As shown in Figure 6, these





compounds differ only in the structure of the terminal

isoprenyl group, which in the case of picrotin is hydrated

to remove the double bond. Picrotoxinin potently inhibits

the GABA A R, whereas picrotin is generally inactive at this

receptor. This behavior correlates well with the relative

systemic toxicities of the two substances. PTX inhibition

of the GABA A R is use dependent (i.e., inhibition reaches

steady-state at a faster rate in the open state) and non-

competitive, and its inhibitory potency is highly sensitiveto TM2 mutations (see below). This combination of prop-

erties has frequently led to PTX being classified as a

channel blocker. However, several lines of evidence (e.g.,

Refs. 277, 296) have firmly established PTX as an alloste-

ric inhibitor of the GABA A R.

In 1992, Pribilla et al. (298) reported that ␣␤-hetero-

meric GlyRs were much less sensitive to PTX inhibition

than were ␣-homomeric GlyRs. This result was indepen-

dent of which ␣-subunit (␣1, ␣2, or ␣3) was investigated.

They also showed that inserting the ␣1-subunit TM2 into

the ␤-subunit bestowed high PTX sensitivity to ␣1␤-

GlyRs. These findings were important for two reasons.

First, by establishing TM2 as a crucial determinant of PTX

sensitivity, they prompted an intensive investigation into

the molecular basis of PTX action in various LGIC mem-

bers (106, 109, 150, 389, 410, 433). Second, they estab-

lished PTX sensitivity as a standard pharmacological tool

for identifying the presence of ␤-subunits in recombinant

and native GlyRs. Although several other compounds also

display subunit sensitivity (see below), there is as yet nobetter tool than PTX for this purpose.

It was subsequently shown that picrotin and picro-

toxinin were equally ef ficacious in inhibiting ␣1-GlyRs

(236). The same study demonstrated that PTX inhibition

was not use dependent and that its inhibition was “com-

petitive,” meaning that its potency decreased as agonist

concentration increased. Both behaviors are uncharacter-

istic of channel blockers. An allosteric mode of action

was confirmed by the findings that the R19ЈL and R19ЈQ

startle disease mutations transformed PTX into an allo-

steric potentiator at low concentrations (Ͻ3 M) and a

FIG. 6. Structures of representative

compounds that exhibit bioactivity at theGlyR.





noncompetitive, slow-onset inhibitor at higher concentra-

tions (236).

A series of studies on the GABA A R, GABA CR, and

GluClR established the 2Ј and 6Ј residues as crucial de-

terminants of PTX sensitivity (106, 109, 150, 389, 410, 433).

A common feature in all of these studies was that a ring of

6Ј threonines was invariably required for high PTX sensi-tivity. Although all GlyR ␣-subunits contain a threonine at

this position, the ␤-subunit contains a phenylalanine. As

anticipated, a range of mutations to T6Ј in the GlyR ␣1-

subunit, including the ␣3 ␤ substitution T6ЈF, greatly di-

minished the inhibitory potency of PTX and related com-

pounds (341, 343, 356). In addition, incorporating a thre-

onine into the ␤-subunit 6Ј position restored high PTX

sensitivity to the ␣1␤-GlyR (341), although a range of

other ␤-subunit 6Ј mutations had no such effect (343).

Does this highly specific requirement mean that T6Ј is the

PTX binding site? Despite a molecular modeling study

finding that PTX can fit into this part of the pore and thatT6Ј hydrogen bonds could plausibly coordinate a PTX

molecule (435), it is premature to draw this conclusion.

The PTX-competitive compound ␣-ethyl-␣-methyl-␥ -

thiobutyrolactone (␣EMTBL; Fig. 6) was found to poten-

tiate glycine responses in ␣1-GlyRs but inhibit them in

␣3-GlyRs (356). The TM2 domains of these subunits are

conserved with the exception of the 2Ј residue: in the

␣1-subunit it is a glycine, but in the ␣3-subunit it is an

alanine. The inhibition seen in the ␣3-GlyR is abolished by

the T6ЈF mutation, although the potentiation seen in the

␣1-GlyR is not affected by this mutation (356). Moreover,

incorporating the ␣1 2Ј residue into the ␣3-subunit (via the A2ЈG mutation) converts ␣EMTBL inhibition into po-

tentiation. To interpret these results in terms of binding

sites, one would have to postulate a site that toggles

between inhibitory and potentiating depending on the

identity of residues at the 2Ј and 6Ј positions. The exis-

tence of two discrete sites (an inhibitory site in the pore

and a potentiating site elsewhere) seems equally implau-

sible. This would require that a mutation that abolishes

the PTX or ␣EMTBL inhibitory sites should simulta-

neously render functional a distant potentiating site.

Thus, although they provide little support for a PTX or

␣EMTBL binding site at T6Ј, the above results arguestrongly for a close allosteric coupling between the 2Ј and

6Ј residues.

The 15Ј residue has also been implicated into this

scheme. When the GlyR ␣1-subunit S15Ј was mutated to a

glutamine or asparagine, inhibition became use depen-

dent and noncompetitive (92). This is similar to the effect

previously seen with R19Ј mutations (236). Because S15Ј

and R19Ј mutations abolished the rapid-onset PTX effect

that is also abolished by T6Ј mutations, it suggests an

allosteric interaction between R19Ј, S15Ј, and T6Ј. Does

this imply that S15Ј is the PTX-binding site? After all,

evidence summarized below strongly implicates S15Ј as

an alcohol and volatile anesthetic binding site.

The current picture regarding the effects of PTX is

complex because mutations to residues at the 2Ј, 6Ј, 15Ј,

and 19Ј positions can each affect the mode or potency of

PTX action. Since all of these mutations have been shown

to exert allosteric effects on PTX binding or effector sites,it is uncertain which, if any, is a PTX contact site. It

appears that an imaginative approach will be required to

convincingly resolve this issue.

Single-channel analysis also reveals complexities in

the actions of PTX. In homomeric ␣-GlyRs, it reduced the

predominant conductance state from ϳ80 to 40 pS, with

the probability of entering the lower conductance state

progressively increasing as the PTX concentration was

raised from 1 to 30 M (217). In contrast, 30 M PTX had

no effect on ␣␤-heteromeric GlyRs, which (perhaps not

coincidentally) also exhibited a 40-pS predominant con-

ductance level (217). Higher (100 M) PTX concentra-tions induced flickery kinetics in both ␣- and ␣␤-GlyRs

(217, 426). These observations support the conclusion

that PTX acts in an allosteric manner.

In summary, experiments to date have revealed an

unusually complex mode of action for PTX. It seems they

brought us little closer to formulating testable hypotheses

concerning the binding site or mechanism of action of this

enigmatic compound.

3. Cyanotriphenylborate

Negatively charged cyanotriphenylborate (CTB; Fig.6) was chosen as a potential GlyR pore blocker due to its

structural similarity with triphenylmethylphosphonium

bromide, a classical nAChR pore blocker. CTB was duly

shown to act in the predicted manner: its inhibition of the

␣1-GlyR was potent (IC50 ϳ1.3 M), use dependent, volt-

age dependent, and noncompetitive (324). Furthermore, it

was not a potent inhibitor of the ␣2-GlyR or ␣3-GlyRs, and

replacing the ␣1-subunit 2Ј residue with the ␣2-subunit

residue (via the G2Ј A mutation) abolished block. To-

gether, these observations provide a strong case for CTB

binding in the pore. However, it is not straightforward to

conclude that CTB binds to the 2Ј glycine, as it also potently blocked an ␣2␤-GlyR in which the ␤-subunit TM2

domain had been entirely replaced by that of the ␣2-

subunit. This paradoxical result demonstrates that CTB

sensitivity can reside in a receptor containing only ala-

nines at the 2Ј position, thereby directly refuting the idea

that the CTB binding site requires 2Ј glycines. One possi-

bility is that regions apart from the TM2 domain may also

contribute to CTB sensitivity (324). Alternatively, ␤-sub-

units may serve the same role as ␣-subunit 2Ј glycines in

creating a favorable geometry for the binding of CTB at

some level in the pore. Progress in characterizing the CTB





mechanism of action is currently limited due to its lack of

commercial availability.

4. Ginkgolides

Isolated from the leaves, roots, and bark of the

Gingko tree, these macrocyclic terpeine compounds are

widely used in herbal medicine. They also share common

structural features with picrotoxinin (170). Ginkgolide B,

which is well known as a platelet activating factor antag-

onist (Fig. 6), is also a specific and potent blocker (IC50

ϳ0.27 M) of glycine-gated currents in dissociated rat

hippocampal pyramidal neurons (192). It is noncompeti-

tive and use dependent, and its inhibitory potency is not

affected by the competitive antagonist strychnine. Impor-

tantly, when applied externally, its blocking ability in-

creased with cell depolarization, as expected for a nega-

tively charged compound binding in the pore (192). These

characteristics establish ginkgolide B as a classical pore

blocker. Its molecular determinants of action and subunitspecificity have yet to be investigated.

More recently, the effects of several ginkgolides were

compared on GlyRs expressed in rat embryonic cortical

neuron slices (170). As these GlyRs were insensitive to

PTX, they may have comprised predominantly ␣2␤-het-

eromers. Ginkgolides B, C, and M were found to be more

potent than ginkgolide A, ginkgolide J, or bilolabide (a

related compound from the same tree). In agreement with

the earlier study (192), ginkgolide B inhibited the GlyR in

a use-dependent, noncompetitive manner and showed

specificity for the GlyR over a GABA A R expressed in the

same tissue (170).

5. Tropisetron and other 5-HT 3 R antagonists

Several 5-HT3R antagonists have potent effects on

the GlyR. The most potent of these compounds are those

which contain tropeine groups (i.e., esters and amides of

3␣-hydroxytropane) and include tropisetron, LY-278,584,

zatosetron, and bemesetron. The muscarinic acetylcho-

line receptor antagonist atropine also belongs in this

structural group. Some representative structures are

given in Figure 6. This review focuses on tropisetron, the

most widely characterized of these compounds.

In 1996, Chesnoy-Marchais found that tropisetron po-tentiated glycine currents in cultured spinal neurons at

low concentrations (0.01–1 M) but caused inhibition at

higher concentrations (66). Its potentiation was mediated

by a leftward shift in the glycine dose-response curve.

Because the potentiation was additive with the potentiat-

ing effects of zinc, ethanol, and propofol (67), tropisetron

appeared to act via a novel mechanism. In radioligand

binding studies on membrane extracts from the spinal

cord and brain stem, tropisetron displaced [3H]strychnine

with a K i of 2 M (240). In addition, 0.1 M tropisetron

increased the ability of glycine to displace [3H]strychnine

(240), suggesting an allosteric enhancing effect on glycine

binding. Turning then to an electrophysiological analysis,

Maksay et al. (242) subsequently failed to detect tropise-

tron potentiation in ␣1- or ␣2-GlyR homomers or in ␣1␤-

or ␣2␤-GlyR heteromers, although the lower apparent

af finity inhibitory effect was observed. In apparent con-

tradiction, Supplisson and Chesnoy-Marchais (358) reported tropisetron potentiation in the ␣1-homomeric and

the ␣1␤- and ␣2␤-heteromeric GlyRs, but not in the ␣2-

homomer where only inhibition was seen (358). The dis-

crepancy was convincingly resolved by the demonstration

that potentiation required a low (EC10) glycine concentra-

tion, whereas higher (EC50) glycine concentrations, as

used by Maksay et al. (242), uncovered only inhibition

(358). The molecular determinants of tropisetron action

have not been elucidated. Because ␤-subunits are needed

to confer tropisetron potentiation to ␣2-subunits (358),

the potentiating binding site may be located at the inter-

face of these subunits.Tropisetron suppresses glycine-gated currents when

applied at high (Ͼ10 M) concentrations to both native

neuronal and recombinant GlyRs. At least in the ␣2-GlyR,

it seems to behave in a noncompetitive manner. The

tropisetron inhibitory potency is modestly affected (IC50

increased by a factor of 2) by the T112A mutation (242), a

mutation that completely abolishes the inhibitory potency

of zinc. The increased tropisetron inhibitory potency of

the ␣2-GlyR is not transferred to the ␣1-GlyR via the A2ЈG

mutation (358), the only TM2 domain residue which is not

conserved between these subunits. However, as previ-

ously seen for CTB and PTX, it may be misleading to infer

locations of binding sites on the basis of site-directedmutations at this position in the pore.

A structure-activity study concluded that the tro-

peine structure itself was required for potentiation (240).

However, atropine, which shares the tropeine moiety,

does not increase glycine displacement of strychnine

binding, although its inhibitory potency is only marginally

weaker than that of tropisetron (240, 242). Thus the tro-

peine moiety appears to be no guarantee of a potentiating

effect. These results are broadly consistent with another

structure-activity analysis, which concluded that an aro-

matic ring, a carbonyl group, and a tropane nitrogen are

required for glycinergic potentiation (70).

6. Ivermectin

Ivermectin (22,23-dihydroavermectin B1a ) is a natu-

rally occurring macrocyclic lactone (Fig. 6) that is widely

used as an antiparasitic agent in agriculture, veterinary

practice, and human medicine (98, 319). Although the

target of its antiparasitic action is believed to be a GluClR

that exists in a number of invertebrate phyla (78, 178), it

also has direct activating or potentiating effects on

GABA A Rs and nAChRs (2, 87, 197, 198). At low (0.03 M)





concentrations, ivermectin potentiates subsaturating gly-

cine responses, but at higher (Ն 0.03 M) concentrations

it irreversibly activates recombinant ␣1- and ␣1␤-GlyRs

(342). Because ivermectin-gated currents have a different

pharmacology to glycine-gated currents, and glycine bind-

ing site mutations do not drastically affect its sensitivity

(342), ivermectin appears to activate the GlyR via a novelmechanism. Apart from cesium (352), ivermectin is the

only non-amino acid agonist of the GlyR to be identified to

date.

7. Alcohols, anesthetics, and inhaled drugs of abuse

Traditionally, anesthetics have been depicted as non-

selective agents that act by partitioning into and disorder-

ing lipid bilayers. However, in recent years it has become

increasingly apparent that specific binding sites on ligand-

gated ion channels are among their major molecular tar-

gets (36, 117, 196, 260, 418). Because alcohol and someanesthetic effects on the GlyR are observed at pharmaco-

logically relevant concentrations, it is possible that at

least part of their acute effects are mediated by this

receptor.

Exposure to pharmacologically relevant (50–100

mM) concentrations of ethanol was first shown by Celen-

tano et al. (59) to produce a persistent increase in the

glycine sensitivity of spinal neurons. Most subsequent

studies on GlyRs natively expressed in neurons have ob-

served similar potentiating effects. Neonatal ventral teg-

mental area neurons appear to constitute an exception to

this rule: ethanol (0.1–10 mM) potentiated, inhibited, or

had no effect on glycine-activated responses in 35, 45, and20%, respectively, of an impressively large sample of these

neurons (422, 423).

Because ethanol potentiation is achieved without dis-

ordering the membrane lipid bilayer (366), it is likely to be

acting via a specific site at the GlyR. When expressed in

Xenopus oocytes, ␣1-GlyRs were more sensitive to etha-

nol than were ␣2-GlyRs (251). This increased sensitivity

was abolished by incorporating the naturally occurring

spasmodic mouse mutation A52S into the ␣1-subunit

(251). However, the ␣1-subunit ethanol sensitivity was

largely lost upon recombinant expression in mammalian

LtkϪ

or HEK293 cells (380). Because the GlyR ␣1-subunitcontains phosphorylation consensus sites, the phosphor-

ylation status of the receptor was considered a possible

cause of this anomaly. Indeed, PKC-mediated phosphory-

lation selectively increases ethanol potentiation of Xeno-

pus oocyte-expressed ␣1-GlyRs while having no effect on

the enhancement induced by the inhalation anesthetic

halothane or the intravenous anesthetic propofol (253).

On the other hand, PKA-mediated phosphorylation was

without effect (3).

The inhalation (or volatile) anesthetic isoflurane was

first shown by Harrison et al. (155) to potentiate recom-

binant ␣2-GlyRs. For both recombinant ␣1-GlyRs and the

native GlyR in medullary neurons, the degree of potenti-

ation increased in the rank order methoxyflurane, sevoflu-

rane Ͻ halothane, isoflurane, enflurane, F3 (97, 250). The

potentiation was induced primarily by a leftward shift in

the glycine EC50 (97). Because the leftward shift is more

pronounced at low glycine EC values (97), the effects of alcohols and volatile anesthetics have generally been in-

vestigated using glycine concentrations that activate 2–5%

of the saturating current magnitude.

In contrast to the GlyR, the GABA CR is inhibited by

both classes of agents (261). By constructing a series of

chimeras between these two receptors, a region of 45

amino acid residues was identified as necessary and suf-

ficient for mediating potentiation by both alcohol and

volatile anesthetics (262). Site-directed mutagenesis of

the nonconserved residues in this region identified S15Ј in

the TM2 domain and A288 in the TM3 domain as crucial

determinants of alcohol and volatile anesthetic sensitivity(262). Because both residues are located towards the

extracellular end of their respective TM segments, it was

hypothesized that these two residues faced each other to

form a pocket to accommodate an alcohol molecule. Ly-

ing within the membrane, this putative water-filled pocket

is thought to be inaccessible from the ion channel pore.

The existence of such a water-filled pocket lined by TM2

and TM3 domains is supported by SCAM data in the

GABA A R and structural evidence from the nAChR (see

sect. III B).

It was already known that alcohol potentiation of the

␣1-GlyR increases with the length of the side chain of a

series of n-alcohols until a cut-off is reached, after whichfurther increases in molecular size decrease alcohol po-

tency (250). Increasing the size of the amino acid side

chain by the S15ЈQ mutation decreased the cutoff from

n ϭ 10 –12 to 7, consistent with the expected reduction in

the volume of the binding pocket (399, 424). The converse

experiment on the GABA CR, in which a smaller side chain

was introduced at the corresponding position, increased

the alcohol size cutoff (399). Using a similar idea, the

molecular volume and hydropathy of the side chain at the

288 position were revealed as crucial determinants of

volatile anesthetic sensitivity (420). Together, these ex-

periments supported the view that S15Ј and A288 mightform the binding pocket for both alcohols and volatile

anesthetics. Alternatively, the mutations might impose

structural changes that perturb the allosteric potentiation

mechanisms of these compounds. Indeed, because these

mutations affect glycine EC50 values (262, 420, 424), this

alternative is a distinct possibility. More direct evidence

for these residues forming a binding pocket came from a

SCAM-type approach (252). Current flux through the ␣1-

GlyR incorporating the S15ЈC mutation was irreversibly

enhanced by the sulfhydryl-containing anesthetic pro-

panethiol under conditions of oxidation by iodine, or





directly by propyl methanethiosulfonate. After modifica-

tion by either compound, the GlyR could no longer be

potentiated by alcohols or anesthetics (252). This consti-

tutes strong evidence that these compounds bind specif-

ically in a pocket lined by S15Ј.

A recent investigation has found that ethanol poten-

tiation of recombinant ␣1-GlyRs was antagonized by in-creased atmospheric pressure (85). Because the in-

creased pressure did not affect the actions of glycine,

strychnine, or zinc, it is likely to exert a selective effect at

the alcohol site. It will be of interest to understand the

molecular basis of this phenomenon.

Recently, several commonly abused inhalants were

investigated in terms of their action at recombinant ␣1-

GlyRs. Toluene, 1,1,1-trichloroethane, trichloroethylene,

and chloroform potentiated ␣1-GlyRs by reducing the

glycine EC50 values (31, 33). Because these compounds

exhibited different patterns of sensitivity to S15Ј muta-

tions than did ethanol and enflurane, and also showedcompetition with these compounds, it was proposed that

their binding sites shared some overlap with the alcohol/

anesthetic binding site (31, 33). However, the possibility

that the respective binding sites may interact allosteri-

cally cannot yet be ruled out.

The gaseous anesthetics nitrous oxide and xenon

weakly potentiate submaximal glycine currents in the

␣1-GlyR at clinically relevant doses (84, 419). Other anes-

thetics including propofol, thiopentone, pentobarbitone,

alphaxalone (a steroid), etomidate, and ketamine also

exert potentiating effects on some GlyR isoforms (35, 84,

250, 295). Because more dramatic effects of all these

compounds are seen at other receptors (36, 116, 118, 196,418), their effects on GlyRs are unlikely to be clinically

relevant.

8. Miscellaneous bioactive compounds

Dihydropyridine antagonists of L-type calcium chan-

nels also inhibit GlyR currents in spinal cord neurons at

micromolar concentrations (69). Their effects are stron-

ger at higher glycine concentrations and increase with

time during glycine application, reminiscent of an open

channel block mechanism. In addition, low (1–5 M)

concentrations of nitrendipine and nicardipine exert po-tentiating effects that were additive with those of zinc,

implying discrete mechanisms of action (69).

The estrogen receptor modulator tamoxifen has re-

cently been shown to have a particularly dramatic poten-

tiating effect on submaximal glycine responses in cul-

tured spinal neurons (68). A 5 M concentration caused a

6.6-fold reduction in the glycine EC50. Several controls

were used to rule out possible effects of tamoxifen on cell

signaling cascades. The magnitude of the potentiation

was increased when tamoxifen was applied to the cells

before glycine application, although maintenance of the

potentiation seen upon glycine application required con-

tinued tamoxifen application (68). The potentiation per-

sisted in the presence of 10 M zinc, implying a discrete

site of action. The same study also noted an apparently

direct potentiating effect of 4 M dideoxyforskolin on

GlyRs, a finding that may be relevant to studies designed

to investigate phosphorylation mechanisms.Clinical concentrations of the neuroprotective drug

riluzole were reported to accelerate the ␣1␤-GlyR desen-

sitization rate while having no effect on the maximal

current magnitude (269). Paradoxically, however, the

time course of currents activated by brief (2 ms) applica-

tions of glycine were prolonged by riluzole (269). Similar

effects were also observed on the GABA A R. The prolon-

gation of the simulated synaptic currents may help ex-

plain the sedative and anticonvulsant side effects of this

drug (95).

Colchicine, a microtubule-depolymerizing reagent,

competitively antagonizes glycine-induced currents withIC50 values of 64 and 324 M for the ␣1- and the ␣2-GlyRs,

respectively. Its effect is instantaneous and independent

of the microtubule depolymerization process (238). Inter-

estingly, colchicine also prevents ethanol potentiation of

GABA A R currents (394, 397), which perhaps provides a

starting point for investigating its mechanism of action.

The tyrosine kinase inhibitor genistein has been

shown to have a direct inhibitory effect on native GlyRs in

neurons isolated from the hypothalamus and VTA (165,

437). This effect is not a pharmacological effect on PTK

activation as it was effective only from the external mem-

brane surface (165). It inhibits in a noncompetitive and

use-dependent manner, suggesting that it binds in the pore.

Reasoning that the pharmacology of the GlyR glycine

site may have structural similarities with the glutamate

(NMDA) receptor glycine site, Schmieden et al. (337) used

a potent NMDA receptor glycine antagonist, 2-carboxy-4-

hydroxyquinoline, as a lead compound for the develop-

ment of novel GlyR antagonists. The most potent com-

pound thus identified, 5,7-dichloro-4-hydroxyquinoline-3-

carboxylic acid, inhibited recombinant ␣1-GlyRs with an

IC50 of 20 M. Although this compound was not active at

the NMDA receptor, the results imply some degree of

similarity in the glycine pharmacophores of the GlyR andNMDA receptors. Another glutamate (KA/AMPA) recep-

tor antagonist, NBQX, has recently been shown to inhibit

recombinant␣1- and ␣2-GlyRs with an IC50 of 4 M (256).

Effects of ginsenosides, the active ingredients of the

Panax ginseng plant, have been investigated on recombi-

nant ␣1-GlyRs. The most active of these compounds, gin-

senoside-Rf, potentiated submaximal glycine-gated cur-

rents with an EC50 of 50 M (282).

Glycine-activated currents in recombinant ␣1- and

␣2-GlyRs were both inhibited by 3-[2-phosphonom-

ethyl[1,1-biphenyl]-3-yl]alanine (PMBA) with an IC50 of





ϳ0.5 M (163). When applied at higher (10 M) concen-

trations, PMBA had differential effects on ␣2- and ␣1-

GlyRs. In the ␣1-GlyR, its main effect was to impose a

rightward shift on the glycine dose-response. However, in

the ␣2-GlyR, it also lowered the Hill coef ficient for glycine

activation (163). Furthermore, preincubation with PMBA

delayed the rate of glycine-gated current activation in the␣2-GlyR but not the ␣1-GlyR. Thus the mode of PMBA

binding or activation may differ between the ␣1- and

␣2-subunits.

Opioid alkaloids have long been known to exhibit

selectivity for the GlyR over the GABA A R (82). The rela-

tive ef ficacy of these compounds in antagonizing glycine-

induced currents in intact spinal neurons is thebaine Ͼ

morphine Ͼ codeine (79, 82), whereas thebaine was by far

the most potent of a series of opioid agonists in displacing

[3H]strychnine binding in spinal cord neurons, with an

IC50 of 1 M (132).

Finally, as reviewed previously (307), a wide range of compounds with structural similarities to various GABA A R

ligands have been investigated in terms of their [3H]strych-

nine displacement potencies. Benzodiazepines and their an-

tagonists were among the most potent compounds thus

identified, with IC50 values of ϳ1–10 M (48, 249, 429).

Interestingly, a recent report has identified a low-af finity

benzodiazepine inhibitory site on ␣2-containing GlyRs (367).

VII. GLYCINE RECEPTOR CHANNELOPATHIES

A. Human Startle Disease

Human hereditary hyperekplexia, or startle disease,

is a rare neurological disorder characterized by an exag-

gerated response to unexpected stimuli (15, 308). The

response is typically accompanied by a temporary but

complete muscular rigidity often resulting in an unpro-

tected fall. This behavior is graphically illustrated in a

published videotape of the bovine form of the disorder

(161). Susceptibility to startle responses is increased by

emotional tension, nervousness, fatigue, and the expecta-

tion of being frightened. Unprotected falls in turn lead to

chronic injuries that are also characteristic of this disor-

der. Symptoms of this disease are present from birth, withinfants also displaying a severe muscular rigidity (or hy-

pertonia) that gradually subsides throughout the first year

of life. Some affected infants die suddenly from lapses in

respiratory function (129). This disorder has a history of

being misdiagnosed as epilepsy, although hyperekplexia

is readily distinguished by an absence of fits and a reten-

tion of consciousness during startle episodes. The symp-

toms are successfully treated by benzodiazepines, with

clonazepam being the current drug of choice (436).

Hyperekplexia is caused by heritable mutations that

reduce the magnitude of glycine-gated chloride currents.

As summarized in Table 1, this is achieved by either

disrupting GlyR surface expression or by reducing the

ability of expressed GlyRs to conduct chloride ions. The

disease mutations thereby impair the ef ficiency of glycin-

ergic neurotransmission in reflex circuits of the spinal

cord and brain stem, thus increasing the general level of

excitability of motor neurons. Patients are apparentlyable to cope with this reduced inhibitory tone during

normal tasks (perhaps due to developmental compensa-

tions, see below), although they are unable to cope with

the increased demand required to dampen strong, unex-

pected excitatory commands.

Genetic linkage analysis of startle disease pedigrees

first localized this disorder to the distal portion of chromo-

some 5q (327), where the GlyR ␣1-subunit gene is found. It

was subsequently shown that an autosomally dominant

form of startle disease was due to either the R271L or R271Q

substitutions in the human ␣1-subunit (346). Although nu-

merous other startle mutations have since been identified,the R271 mutations seem to be the most common cause of

this disorder. When incorporated into recombinantly ex-

pressed GlyRs, these mutations caused a reduced glycine

sensitivity and single-channel conductance (208, 306). Other

autosomal dominant startle mutations, including Y279C,

K276E, Q266H, and P250T, have generally similar effects

(Table 1). In addition, the P250T mutation causes an en-

hanced desensitization rate (334), which may also contrib-

ute to the disease phenotype. As discussed previously in this

review, each of these mutations acts by impairing the recep-

tor activation mechanism, and closer analysis of the muta-

tion phenotypes has provided valuable insights into the

structural basis of GlyR function. The effect of the autoso-mally dominant V260M and S270T startle disease mutations

(88, 210) has yet to be characterized.

Autosomally recessive forms of startle disease have

been described in sporadic cases, generally in the off-

spring of consanguineous parents. One such patient ex-

hibited homozygosity for a point mutation, I244N (312), in

the ␣1-subunit. When incorporated into recombinant ␣1-

homomeric GlyRs, this mutation resulted in a reduced

glycine current magnitude, a reduced glycine sensitivity,

and an enhanced desensitization rate (237). Two reces-

sive forms of startle disease have also been described that

must have resulted in the complete nonexpression of theGlyR ␣1-subunit. In one case, the mutation caused a ho-

mozygous deletion encompassing exons 1– 6 (53),

whereas the other involved a base pair deletion resulting

in a frameshift and a premature stop codon prior to the

TM1 domain (315). Despite the complete absence of ␣1-

subunit expression, the symptoms experienced by these

patients were no more severe than in those where the

␣1-subunit function was only mildly impaired. Another

recently identified ␣1-subunit recessive point mutation,

S231R, caused a reduced membrane insertion of func-

tional receptors (166).





Compound heterozygous forms of startle diseasehave also been described. In one case, two distinct reces-

sive ␣1-subunit mutations, R252H and R392H, caused

startle disease when present in different alleles (384).

However, patients possessing either single mutant allele

were healthy. Consistent with this observation, coexpres-

sion of the two mutant subunits resulted in a reduced

surface expression of functional GlyRs, whereas the indi-

vidual mutations had no effect (311). The recessive

M147V and G342S ␣1-subunit mutations have also been

identified in patients exhibiting startle disease symptoms

(315). These mutations are presumed to result in com-

pound heterozygous forms of startle disease, but if this isthe case their partner mutations remain to be identified.

Finally, compound heterozygous mutations have recently

been identified in the ␤-subunit. A ␤-subunit missense

mutation (G229D) and a splice site mutation (resulting in

the excision of exon 5) occurred simultaneously in a

compound heterozygote with a transient startle disease

phenotype (314). The G229D mutation alone induced a

modest (4-fold) decrease in the glycine sensitivity of re-

combinant ␣1␤-GlyRs (314), whereas the removal of exon

5 would presumably have precluded the expression of

functional ␤-subunits.

To date, no startle mutations have been identified inthe human ␣2- or ␣3-subunits. It remains to be determined

for any species whether expression of these subunits is

upregulated to compensate for the loss of ␣1-subunit

expression or function. The effectiveness of clonazepam

in treating startle disease is presumably due to its poten-

tiation of the GABA A R. Indeed, evidence from spastic

mice (26) (135) and myoclonic cattle (233) suggests that

spinal cord GABAergic neurotransmission may be up-

regulated during development in compensation for the

loss of glycinergic tone.

It would not be surprising if startle syndromes re-

sulted from mutations that disrupt the function of other proteins involved in the formation, maintenance, and

function of glycinergic synapses. Indeed, a hereditary mu-

tation in the GlyR clustering protein gephyrin results in a

hyperekplexia phenotype in humans (313) and targeted

deletion of the glycine transporter subtype 2 gene pro-

duces a startle phenotype in mice (133).

B. Murine Startle Syndromes

Three naturally occurring murine startle syndromes

have been identified to date (Table 1). The symptoms of

TABLE 1. GlyR mutations underlying startle syndromes in various species

Mutation and Subunit Inheritance Mode Effect on GlyR Function Reference Nos.

Human forms

␣1 P250T Autosomal dominant Reduced single-channel conductance, reduced glycinesensitivity, increased desensitization rate

50, 334

␣1 V260M Autosomal dominant As yet unknown 88␣1 Q266H Autosomal dominant Reduced open probability, reduced glycine sensitivity 263, 271␣1 S270T Autosomal dominant As yet unknown 210

␣1 R271L/Q Autosomal dominant Reduced glycine sensitivity, reduced single-channel

conductance

208, 306, 327, 346

␣1 K276E Autosomal dominant Reduced glycine sensitivity, reduced open probability 101, 228, 237

␣1 Y279C Autosomal dominant, variable

penetrance

Reduced glycine sensitivity, reduced whole cell current

magnitude

205, 237, 345

␣1 I244N Autosomal recessive Reduced glycine sensitivity, reduced whole cell currentmagnitude, increased desensitization rate

237, 312

␣1 Deletion of exons 1-6 Autosomal recessive Presumed nonfunctional 53␣1 S231R Autosomal recessive Reduced membrane insertion 166

␣1 Stop codon at Y202 Autosomal recessive Reduced surface expression, possible heterozygosity

with ␣1 V147M

315

␣1 G342S Compound heterozygous? No effect of individual mutation, possibleheterozygosity with other mutations

315

␣1 R252H ϩ ␣1 R392H Compound heterozygous Reduced membrane insertion 311, 384␤ G229D ϩ ␤ exon 5 loss Compound heterozygous Reduced glycine sensitivity, reduced surface expression 314

Murine forms

␤ Line-1 intronic insertion Autosomal recessive( Spastic)

Reduced surface expression 156, 187, 275

␣1 A52S Autosomal recessive

( Spasmodic)

Reduced glycine sensitivity 335

␣1 Stop codon Autosomal recessive(Oscillator )

Reduced surface expression 54

Bovine form

␣1 Stop codon Autosomal recessive( Myoclonus)

Reduced surface expression 147, 294





each are largely similar to those observed in human hy-

perekplexia (308). An exception is that mouse startle

symptoms commence at around the 20th postnatal day,

corresponding to the time when the replacement of ␣2-

homomers by ␣1␤-heteromers is complete. The most

thoroughly characterized murine syndrome is the spastic

mouse, which was first identified in the 1960s (255). Thisautosomally recessive disorder is characterized by a re-

duction in the number of expressed GlyR ␣1-subunits,

although the function of the expressed receptors is nor-

mal (26, 28). The spastic phenotype was found to be

caused by the insertion of a 7.1 kb Line-1 repeating ele-

ment into intron 5 of the ␤-subunit (187, 275). This ele-

ment leads to aberrant splicing of the ␤-subunit tran-

scripts resulting in an accumulation of prematurely ter-

minated protein and a concomitant reduction in the

surface expression of ␤-subunits (187, 275). Because

␤-subunits are required to anchor ␣1-subunits to synaptic

scaffolding proteins, the disease phenotype is presumablycaused by a loss in the ef ficiency of GlyR recruitment to

postsynaptic densities. Introduction of low levels of a

wild-type ␤-subunit transgene effectively reverses this

disease phenotype (156).

As expected, the magnitudes of glycinergic IPSPs in

motor and sensory spinal neurons of spastic mice were

significantly reduced relative to wild-type controls (135,

386). However, the same two studies observed contradic-

tory effects on the size of GABAergic IPSPs in the same

neurons. Graham et al. (135), who measured the magni-

tude of spontaneous mini-IPSPs in dorsal horn sensory

neurons, reported an increase in GABAergic IPSP magni-

tude. On the other hand, Von Wegerer et al. (386), whorecorded electrically stimulated IPSPs in ventral horn

motor neurons, reported a reduction in GABAergic IPSP

magnitude. The difference may relate to the identity of the

neurons studied or to the different IPSPs (evoked versus

spontaneous) measured.

The spasmodic mouse contains the homozygous

point mutation A52S in the ␣1-subunit. This mutation

results in a moderate (6-fold) reduction in glycine sensi-

tivity (326, 335). Spinal inhibitory neurotransmission has

yet to be characterized in this mouse. An allelic variant of

spasmodic, termed oscillator , is the only lethal form of

startle disease identified to date. This syndrome is causedby a frameshift mutation in the TM3-TM4 domain result-

ing in the translation of an incomplete form of the ␣1-

subunit protein (54). With the assumption that the major-

ity of spinal glycinergic synapses comprise ␣1␤-hetero-

mers by postnatal day 20, homozygous oscillator mice

should have virtually no glycinergic tone. Consistent with

this, membranes isolated from oscillator homozygote spi-

nal cords display a 90% reduction in strychnine binding,

indicating a drastic loss in the functional expression of

the ␣1-subunit (54). Surprisingly, however, the magnitude

of strychnine-sensitive synaptic currents was found to be

reduced by only 50% in dorsal horn sensory neurons of

mice homozygous for this mutation (135). It would be of

interest to examine the pharmacology of the currents that

remain: is it possible that the strychnine-insensitive ␣2*-

subunit is upregulated to compensate for the loss of ␣1-

subunits?

It is noteworthy that lethality does not result fromhuman and bovine startle mutations that act similarly to

completely preclude the functional expression of ␣1-sub-

units (53, 294, 315). A possible reason for the difference is

that the ␣2 3 ␣1␤ subunit switch in the mouse occurs

after birth resulting in an inability to feed. In humans and

cattle, the switch is likely to occur before birth, thereby

not affecting the ability to feed, and affording time for the

development of synaptic adaptations before parturition.

Two transgenic mice strains have been generated

with a view to developing an animal model of human

hyperekplexia. The first approach mimicked the spastic

mouse mutation by engineering a transposon insertioninto the ␤-subunit (29). Homozygous mice displayed a

startle phenotype that resembled human hyperekplexia

(30). The second approach incorporated the dominant

human ␣1-subunit R271Q mutation into transgenic mice.

Transgenic mice that were heterozygous for the R271Q

human startle mutation displayed a pronounced startle

phenotype, and mice homozygous for the mutation were

not viable (30). Knock-in mice bearing the ␣1-subunit

S267Q mutation (which eliminates the alcohol binding

site and reduces peak current magnitude) displayed a

reduced sensitivity to alcohol and an oscillator -type phe-

notype (111, 112). Finally, as noted above, knock-out of

the glycine transporter subtype-2 gene produces a lethaloscillator -type phenotype (133).

C. Bovine Myoclonus

A congential recessive startle syndrome, called my-

oclonus, has been identified in Poll Hereford cattle (160).

This disorder was recently shown to be due to a single

base pair deletion in the ␣1-subunit gene, leading to a

frameshift and a premature stop codon before TM1 (294).

As would be expected, this mutation induced a dramatic

reduction in the surface expression of functional GlyRs

(147). A similar syndrome may also exist in Peruvian Paso

horses (148).

VIII. OUTLOOK

GlyRs have important roles in a variety of physiolog-

ical processes, especially in mediating inhibitory neuro-

transmission in the spinal cord and brain stem. Although

recent progress in understanding the molecular func-

tional architecture of these receptors has been rapid,

many gaps remain in a number of critical areas. The





following are considered to be among the most pressing

research priorities at the present time.

1) A high-resolution structure of the entire GlyR com-

plex will permit the design of much more precise exper-

iments to understand receptor structure and function.

Unfortunately, however, crystal structures are notori-

ously dif ficult to obtain for membrane-spanning proteins,and complete structures have yet to be resolved for any

LGIC member.

2) It is essential to establish the exposure pattern of

residues in TM2 and in the pore selectivity filter of the

GlyR ␣- and ␤-subunits. This is a prerequisite for under-

standing the ion-permeation and channel-opening mecha-

nisms. It is also essential to probe the surface electro-

static potential in the pore selectivity filter to further our

understanding of the ionic charge discrimination mecha-

nism.

3) Because binding sites are located at subunit inter-

faces, it is necessary to resolve the GlyR subunit stoichi-ometry and arrangement so that the number and type of

subunit interfaces can be defined. This is an important

first step toward resolving the structural and functional

basis of ligand binding.

4) There are large gaps in our knowledge of glycine

binding mechanisms. In particular, the role of the ␣-sub-

unit complementary binding domains in coordinating gly-

cine needs to be investigated. This is a prerequisite for the

structural modeling of the agonist binding pocket.

5) The role of ␤-subunit in channel in agonist binding

and channel activation has received scant attention.

Again, this information is necessary for the structural

modeling of ligand binding sites and the understanding of activation mechanisms.

6) The modeling of GlyR kinetics remains controver-

sial. Because kinetic models are crucial for understanding

and predicting receptor behavior, it is hoped that future

studies will carefully readdress this situation.

7 ) The diversity of GlyR subtypes at least partially

underlies the diversity in glycinergic neurotransmission

properties throughout the central nervous system (219).

Understanding the GlyR structural variations that under-

lie this synaptic functional diversity is an important ques-

tion for future research.

8) The role of ␣2-homomeric GlyRs in embryonicneurons remains to be clarified.

9) Extrasynaptic GlyRs are present on many central

nervous system neurons. In addition, GlyRs have been

found in a number of nonneuronal tissues. The physiolog-

ical roles of these GlyRs need to be investigated in more

detail.

10) The therapeutic possibilities of GlyR modulatory

agents warrant further investigation. As noted in a recent

review (215), the fact that GlyRs are involved in motor

reflex circuits and nociceptive sensory pathways suggests

that GlyR modulators could have therapeutic potential as

analgesics and muscle relaxants. This review describes

the effects of a number of molecules with potential to be

lead compounds for the development of such therapeu-

tics. Further therapeutic possibilities may emerge as the

roles of extrasynaptic and nonneuronal GlyRs are char-

acterized in more detail.

I gratefully acknowledge the patience of my laboratory

members during the writing of this review. Professor Peter

Barry and the anonymous reviewers are acknowledged for pro-

viding valuable insights and helpful suggestions. Dr. Brett Cro-

mer and Prof. Michael Parker are thanked for kindly providing

the unpublished image in Figure 3 B.

Research in the author ’s laboratory is funded by the Aus-

tralian Research Council and the National Health and Medical

Research Council of Australia.

Address for reprint requests and other correspondence: J.

W. Lynch, School of Biomedical Sciences, Univ. of Queensland,

Brisbane QLD 4072, Australia (E-mail: [email protected]).

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1 GlycineαThe Basic Property of Lys385 Is Important for Potentiation of the Human

including high resolution figures, can be found at:Updated information and services

http://physrev.physiology.org/content/84/4/1051.full.html

can be found at: Physiological ReviewsaboutAdditional material and information

http://www.the-aps.org/publications/prv

This infomation is current as of February 4, 2012.


http://www.jbc.org/content/286/40/35129.full.pdf


http://www.jbc.org/content/286/40/35129.full.html

http://www.jbc.org/content/286/40/35129.abstract.html





http://jpet.aspetjournals.org/content/339/2/386.full.pdf


http://jpet.aspetjournals.org/content/339/2/386.full.html

http://jpet.aspetjournals.org/content/339/2/386.abstract.html





http://jnnp.bmj.com/content/82/12/1399.full.pdf


http://jnnp.bmj.com/content/82/12/1399.full.html

http://jnnp.bmj.com/content/82/12/1399.abstract.html



























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