chapter 21 gap junctions as electrical synapses

Chapter 21

Gap Junctions as Electrical Synapses

Juan Mauricio Garre and Michael V.L. Bennett

Abstract Gap junctions form between many cell types. Between neurons, they

constitute one class of electrical synapses. Gap junctions are aggregates of

membrane channels between the conjoined cells and in mammals they are

comprised of connexins, which are encoded by a gene family that has 21

members in humans. Each of the coupled cells contributes a hemichannel to

each cell–cell channel. Channel turnover can occur within hours, or channels

may last a lifetime. Not all connexins will form channels with every other

connexin, and connexin compatibility is one limit on junction formation.

Other mechanisms including cell attachment and recognition molecules con-

tribute to specificity of gap junction formation. Electrical synapses are char-

acterized by specificity, but mistakes, i.e., inappropriate connections, are some-

times made. Pannexins/innexins form gap junctions in invertebrates, but

apparently only hemichannels in mammals.

Keywords Gap junction � Connexin � Pannexin � Innexin � Electrical coupling �Dye coupling � Synchronization � Synaptic delay

21.1 Introduction

In this chapter we consider formation of gap junctions, which are electrical

synapses when they are between neurons and are also common between other

cell types, both electrically excitable and inexcitable. Gap junctions are aggre-

gates of cell–cell channels that connect the cytoplasm of the coupled cells. They

mediate electrical transmission, which is the flow of current, largely carried by

K+ ions and driven by the potential difference between cells. They also allow

movement of small molecules, charged and uncharged, by simple diffusion.

M.V.L. Bennett (*)Dominick P Purpura Department of Neuroscience, Albert Einstein Collegeof Medicine, Bronx, NY 10804, USAe-mail: [email protected]

M. Hortsch, H. Umemori (eds.), The Sticky Synapse,DOI 10.1007/978-0-387-92708-4_21, � Springer ScienceþBusiness Media, LLC 2009

423

Gap junctions in vertebrates are formed by connexins, a protein family encodedby 21 genes in humans (Sohl and Willecke 2004). A brief discussion of inverte-brate gap junction forming proteins, innexins, and their vertebrate homologs,pannexins, appears at the end of this chapter; for now gap junctions refers onlyto those comprised of connexins. A single gap junction channel is composed oftwo connexin hexamers, one in each of the apposed membranes that are dockedtogether across the eponymic gap between the cells; there is essentially no leakfrom the channel to the extracellular space of the gap. The hexamers are calledhemichannels or connexons.

Not uncommonly, cells express multiple connexins, and a connexin hexamercan be homomeric, i.e., made of a single connexin, or heteromeric, i.e., made ofmore than one connexin. A junction can be homotypic, as between hemichannelsof the same kind, or heterotypic, when formed by different connexins in the twohemichannels. A further nomenclatural nicety is whether junctions are betweencells of the same or different kind, homocellular or heterocellular. Another pair ofterms, homologous and heterologous, is sometimes used for these latter junc-tions. This nomenclature is suboptimal, because ‘‘homologous’’ is generally usedto refer to molecules or structures that have a common evolutionary origin.

In this chapter we will not consider other forms of electrical interaction,which include the electrical inhibitory synapse on the Mauthner cell of teleostsand mutual excitation by electrical coupling across extracellular space. Theformer has a complex anatomy involving several cell types (Nakajima 1974),and the latter is generally an unspecialized area of apposition with little mor-phological specialization (see Bennett 1972, 1977).

21.2 Life History of a Gap Junction

To form a gap junction, each cell must express a connexin capable of making achannel with a connexin in the other cell. Pairings that have been shown toallow or not to allow junction formation are shown in Table 21.1. This table hasmany blank entries, and for connexins that are expressed in few cell types, mostpossible heterotypic combinations are not physiologically relevant.

Connexins are cotranslationally inserted into membrane of the endoplasmicreticulum (ER) and can be assembled into hexamers in the ER (Koval 2006) orin a post-Golgi compartment (Musil and Goodenough 1993). [Cx26 can besynthesized in the absence of membrane and inserted into membranes post-translationally (Zhang et al. 1996, Ahmad and Evans 2002).] No connexin isknown to be glycosylated, so this function of the Golgi complex is not required.Newly synthesized hexamers are transported to the plasma membrane in vesiclesthat are ultimately inserted into the membrane, the luminal aspect of the hemi-channel in the vesicle now facing the external milieu (Fig. 21.1). Trafficking maybe along microtubules and vectorial for delivery close to tight junctions (Shawet al. 2007). [Cx26 may again be an exception as it is transported along actin

424 J.M. Garre and M.V.L. Bennett

filaments (Thomas et al. 2001).] Many epithelia are characterized by apical and

basolateral faces with different molecular compositions, and hemichannel inser-

tion can be polarized. Alternatively, at least in cell lines and possibly unpolarized

cells such as fibroblasts and many types of astrocytes, insertion may not be

Table 21.1 Connexin compatibility, or which (homomeric) connexin hemichannels will formgap junctions with which?

Cx 26 30 30.2 32 36 37 40 43 45 46 47 50

26 + + + � � � � + + +

30 + + + � + + + +

30.2 + + + + +

32 + + + + � � � � � + + +

36 � � � + + � + + � + �37 � + � + + + + + + �40 � + + � � + + � + � �43 � + + � + + � + + + + �45 + + + � + + + + + � +

46 + + � + � + � + +

47 + + + + +

50 + + � � � � + +

In most cases, recombinant connexins were expressed in cell lines. Although there are anumber of studies of structural requirements for compatibility, there is no clear relationbetween connexin group and compatibility. +: junctions do form. �: junctions do notform. blank: not examined. (from Bukauskas 2001 and his unpublished work).

(enlarged)

insert and diffuse to find partnerin an apposed membrane

Lumen becomesextracellular

ER

Golgi

Fig. 21.1 Connexin hemichannels are cotranslationally inserted into membrane of the endo-plasmic reticulum and assembled into hemichannels, either in the endoplasmic reticulum or ina post-Golgi compartment. Vesicles with hemichannels are transported to the plasma mem-brane and inserted. Hemichannels can then diffuse laterally to find and dock with hemichan-nels in an apposed membrane. A common site of docking is along the edge of a preexistingjunction where the membranes closely approach one another (diagram by Jorge E Contrerasmodified from Bennett 2008)

21 Gap Junctions as Electrical Synapses 425

particularly localized. Once inserted hemichannels appear free to diffuse in thesurface membrane. However, some connexins have PDZ and other bindingmotifs, which might restrict them to specific cell regions (Giepmans 2004).Furthermore, participation in a gap junction leads to reduced mobility becauseof increased mass and attachment to the apposed cell. If there is a preexisting gapjunction, the hemichannels in non-junctionalmembrane can diffuse to the edge ofthe junction where the membranes are closely apposed and they are able to dockwith a hemichannel in the apposedmembrane. The intercellular gap of nominally20 nm contains cell adhesion molecules, glycoproteins, and other molecules thatare anchored in the membranes lining the gap. This space is much too large forconnexin hemichannels on either side to interact with each other.

Timed labeling of new channels shows that they are added at the edge ofexisting gap junctions (Gaietta et al. 2002, Segretain and Falk 2004). Moreover,in a gap junction that remains of a constant size, channels near the center of thegap junction are internalized at a rate equal to that at which they are added at theedge. The internalization process is remarkable. The entire junction thickness,including both membranes and channels, where each cell contributes a hemi-channel, are taken up into one or the other cell, along with a little cytoplasm fromthe donor (or loser) cell (Fig. 21.2). In this process the surface membrane of eachcell is broken and resealed, and the corresponding membranes in the internalizedjunction are also resealed, and the cytoplasm in the vesicle also retains its contents(see Gumpert et al. 2008). Subsequently, the internalized vesicles are transportedto lysosomes, where they are degraded. There is no indication that gap junctioncomponents are recycled (other than by reentry into cell metabolism).

Although cell–cell channels are not reused, unapposed hemichannels in thesurface membrane can be internalized and then reinserted as part of the con-stitutive recycling of surface membrane (VanSlyke and Musil 2005). Reduceddegradation of the internalized hemichannels by a variety of metabolic stressorsincreases surface expression and formation of gap junctions, possibly by simplemass action.

In some gap junctions formed of Cx32 or Cx43, turnover is surprisingly rapid,a matter of a few hours (Gaietta et al. 2002). At the opposite extreme, gapjunctions in the lens nucleus, which are formed of Cx46 and Cx50 (Zampighiet al. 2000), cannot turn over, as there is no capacity for protein synthesis in themature lens fibers, and the junctions last a lifetime. The significance of the rapidturnover is obscure, but does allow changes in cell–cell coupling by alterations inthe rate of internalization over reasonable time frames. For most connexins,turnover times have not been determined, and we do not know the extent towhich they vary among different cells and tissues expressing the same connexins.

Until relatively recently, it was thought that hemichannels in the surfacemembrane would not open until docking with another hemichannel in anapposed membrane; the high permeability suggested by the permeability ofcell–cell channels would lead to excess Ca2+ and Na+ influx and loss ofmetabolites. Now it is clear that under certain conditions hemichannels canopen, although with low probability, and that open hemichannels may mediate


autocrine and paracrine cell signaling, as well as an increase in cell stress(Bennett et al. 2003)

21.3 The First Opening

Using dual whole cell patch clamp recording it is possible to detect the first gapjunction channel opening when two cells are brought together. If cells that donot express connexins are transfected with Cx43-EGFP (Cx43 with enhanced

green fluorescent protein attached to its C-terminal), one can visualize theformation of gap junctions (Bukauskas et al. 2000). Not surprisingly, if thereis a large fluorescent aggregate of gap junction channels (or a junctional plaque)between the cells, there is a large junctional conductance between them, and if

cell 2cell 1

The evagination of cell 2has pinched off.

cell 2cell 1

An evagination of cell 2 with the junction protrudes

into cell 1.

cell 2cell 1

Cell1 has endocytosedthe evagination of cell 2

and the junction.

cell 2cell 1

gap junction

intercellular space

a b

dc

Fig. 21.2 Steps during the internalization of a gap junction. Internalization of small centralareas of a gap junction is the more common mode, but this diagram clarifies the breaking andresealing of the two plasma membranes. The internalized junction is destined for lysosomaldegradation (modified from Bennett 2009)


there is no apparent junction, there is no coupling. Now one asks how big ajunction must be for the first channel to open. One might predict that no visibleaggregate of channels would be necessary, if an isolated channel could open.Surprisingly, an aggregate of several hundred channels must be present for thefirst channel to open. [The number of channels can be estimated from the totalfluorescence of the aggregate. Whether these aggregates are docked hemichan-nels is uncertain (see Preus et al. 1981).] Moreover, as the plaque increases insize, junctional conductance increases, but only a small percentage of thechannels are open at any one time. It is unclear, whether the channels that areopening (and closing) are a subpopulation opening with a high probability or alarger fraction opening with a low probability. [Estimates from gap junctions atthe club endings on the Mauthner cell also suggest that only a small fraction ofthe channels are open at any one time (Tuttle et al. 1986, Pereda et al. 2004).]

We do not know the mechanism(s) underlying the requirement for manychannels (or hemichannels) in the apposed membranes to allow the opening ofeven a single channel. Candidates are tension along the axis of the channel orlateral compression associated with forces in the surface membrane causing thechannels to aggregate. The glycocalyx molecules that must be pushed aside toallow the hemichannels in the apposed membranes to make contact provide aforce analogous to surface tension that would tend to cause the channels toaggregate (the Cheerio1 effect, Fig. 21.3). The compressive force aggregatingthe channels may be required for channel opening.

The dependence on multiple channels for a single one to open would notappear to apply to very small junctions such as those formed of strings ofparticles in the retina (Raviola and Gilula 1973) or to isolated, unapposedhemichannels in the surface but not at junctions (Bennett et al. 2003). Smalljunctions containing only tens of particles have also been described (e.g., Rashet al. 2007b). Moreover, the question arises of how the first junction is formed.Cells may be able to put out filopodia that can overcome the structural barrierprovided by adhesion molecules (Yamane et al. 1999).

Tight junctions are another site where membranes approach closely enoughfor gap junction hemichannels to touch each other. At these structures theextracellular space is completely occluded, and aggregates of gap junction chan-nels are often found alongside tight junction strands. Facilitated cell–cell channelformation at tight junctions may account for targeted transport of hemichannelcontaining vesicles to regions where there are tight junctions (Shaw et al. 2007).

21.4 Gap Junctions as Sites of Attachment

Using appropriate detergents gap junctions can be isolated more or less intact.Thus it appears that the two cells are mechanically held together by gapjunctions. Low Ca2+ solutions that allow cell dissociation by disruptingCa2+-dependent attachments at desmosomal and adherens junctions do not


separate gap junctions. When tissues are mechanically dissociated in low Ca2+

solutions, intact gap junctions go with one or the other cell, often with a vesicle

of membrane from the previously coupled cell (Mazet et al. 1985). Isolated

junctions can have one membrane stripped away with the probe of an atomic

force microscope to expose the extracellular aspect of the hemichannels in the

other membrane (Muller et al. 2002), so a potential plane of cleavage does

appear to be present at the center of the channels. Conventional methods of cell

dissociation do not separate the channels at this site. Although gap junctions do

not have the cytoskeletal connections of desmosomes, interactions with cytos-

keletal elements such as ZO-1 do occur (Thomas et al. 2002, Giepmans 2004). In

at least some cases gap junction internalization is mediated by clathrin (Gum-

pert et al. 2008), which implies a mechanical connection, although possibly a

weak one compared to that of the classical adhering junctions.Cx43 is necessary for neuroblast migration along radial glia. However, this

cell migration appears not to require the formation of patent intercellular

channels, and a mechanical function for Cx43 was inferred (Elias et al. 2007).

More fluid lifted up at the edge of a clusterof Cheerios than where they abut.

aqueouschannel

connexon connexin monomer

plasma membranes

2 – 4 nm gap

intercellular space

A

B

Fig. 21.3 A mechanism for clustering of gap junction channels, the Cheerio1 effect. (A) Themembranes of the coupled cells are much closer at a gap junction than the normal separationbetween them, a space into which cell adhesion and glycocalyx molecules extend. Energy isrequired to push these molecules aside to narrow the extracellular space to a separation acrosswhich gap junction channels can form. The energy requirement is less if channels are clustered.(B) Similarly, Cheerios1 cluster on the surface of milk or water because less fluid is raisedwhere the Cheerios1 are in contact than at the outer edge of a cluster (gap junction diagramfrom Wikipedia)


Gap junctions also have a role in invasion of glioblastoma cells. This functionmay be adhesive or may be mediated by some other aspect of gap junctionalcommunication (Oliveira et al. 2005, Cotrina et al. 2008). The adhesive roles ofgap junctions are reviewed in more detail by Prochnow and Dermietzel (2008).

21.5 Specificity of Junction Formation in the Central

Nervous System

For cells to form heterocellular gap junctions they must express connexins thatwill form gap junctions with each other (Table 21.1). In the central nervoussystem, neurons, astrocytes, and oligodendrocytes all live together and fre-quently contact one another. Each cell type expresses different connexins,which to some extent will limit the formation of heterocellular junctions.Astrocytes express Cx26, Cx30, and Cx43 and oligodendrocytes express Cx29,Cx32, and Cx47 (Altevogt and Paul 2004, Li et al. 2004, Nagy et al. 2004,Orthmann-Murphy et al. 2007). Astrocytes are extensively coupled to eachother, primarily by Cx43 junctions. However, subpopulations in the CNS canform restricted networks implying specificity mechanisms in gap junction for-mation between astrocytes (Houades et al. 2008). Oligodendrocytes only rarelycouple to each other, but do couple to astrocytes through (Cx26 and/or Cx30)/Cx32 and Cx43/Cx47 junctions on their cell bodies (Nagy et al. 2003). Cx29 isapposed to the axonal membrane in the paranodal region and probably doesnot form gap junctions (Kleopa et al. 2004).

Neurons express primarily Cx36 and variably Cx45 (and Cx57 in the hor-izontal cells of the retina) (Nagy et al. 2004). A new neuronal connexin is Cx30.2that is found in inhibitory interneurons, often coexpressed with Cx36 (Kreuz-berg et al. 2008), and also in the atrioventricular conduction system of the heart(Bukauskas et al. 2006). Although Cx36 will form gap junctions with Cx43,Cx45, and Cx47 in expression systems, it does not appear to do so in the CNS,and there is no evidence of glial/neuronal junctions in the adult nervous system(Rash et al. 2000, Nagy et al. 2004, Rash et al. 2005). Cx30.2 is compatible withCx40, Cx43, and Cx45 (Table 21.1), but as yet there are no data on its coloca-lization with other connexins at gap junctions in the nervous system.

21.6 Specificity of Gap Junction Formation Between Neurons

Connexin incompatibility clearly does not account for the absence of glial/neuronal coupling. One must appeal to other cell adhesion and recognitionmolecules, such as those that have demonstrated roles in the specificity offormation of chemical synapses. For a description of these other adhesivemolecules see Chapters 7–20. An early example of the importance of adhesionmolecules for junction formation was the greatly increased incidence of gap


junctions between mouse sarcoma cells after transfection with a cell adhesionmolecule (Mege et al. 1988). Other adhesion molecules are implicated in gapjunction formation in studies of other non-neuronal tissues (Meyer et al. 1992,Prowse et al. 1997, Yamane et al. 1999, Wei et al. 2005). Here we cite a fewexamples of gap junction synapses, where specific contacts are made and gapjunction formation is not a simple outcome of cell proximity. As for couplingbetween glial cells and the absence of coupling between glia and neurons,specificity is likely to be determined by the same or similar molecules as thosethat determine the formation of chemical synapses. (There remains the conun-drum that these mechanisms of adhesion usually do not get the membranes inclose enough proximity to form gap junctions.)

There are two main classes of electrical synapses between neurons, gapjunctions at axodendritic or axosomatic contacts, and gap junctions betweendendrites or somata (Fig. 21.4A, B). Coupling of dendrites and cell bodies tendssynchronize impulse activity of the coupled cells. Axosomatic and axodendriticgap junctions can shorten propagation time between cells, particularly in coldblooded animals where synaptic delays tend to be longer. These junctions canalso couple and synchronize the postsynaptic cells, if the axons have shortbranches forming gap junctions on multiple cells (Fig. 21.4C).

Inhibitory interneurons are commonly coupled by dendrodendritic junctions(Gibson et al. 1999, Fukuda and Kosaka 2000,Mann-Metzer and Yarom 2000,

e e

e

eee

e

e

i

i

i

Fig. 21.4 Types of synapse. (A) Axosomatic synapses. Cell 1 forms a chemical synapse on cell3. Arrows indicate current flow if the synapse is excitatory (e) or inhibitory (i). Cell 2 forms anexcitatory electrical synapse on cell 3. During the presynaptic impulse Na+ flows into theterminal andK+, the main charge carrier in the cytoplasm, flows through gap junction(s) intothe postsynaptic cell. The initial segment, where the impulse arises, has difficultydistinguishing the mode of excitatory transmission. (B) Dendrodendritic synapse mediatingelectrotonic coupling and synchronous activity of neuronal somata. If the arrow indicates thedirection of current flow, the cell at the arrow head is excited (e) and the cell at the tail isinhibited (i); thus, the synapses are both excitatory and inhibitory. (C) Coupling of cell bodiesby way of presynaptic fibers can serve the same synchronizing function as dendrodendriticjunctions (modified from Bennett 1972)


Bennett and Zukin 2004, Connors and Long 2004, Cruikshank et al. 2005).

Coupling is not very close, but is sufficient to loosely synchronize cell

firing. This activity is implicated in generation of gamma rhythms

(30–60 Hz oscillations in the electroencephalogram that are implicated in

perceptual processes, see Bartos et al. 2007). In cortical areas, there are

multiple types of interneurons, e.g., fast spiking (FS) and low-threshold

spiking (LTS). Each class of neurons tends to be coupled to like neurons,

with a small fraction being coupled across classes. These cases may arise

from the failure of the specificity mechanisms. Possible developmental

errors have been described in the retina, where horizontal cells are coupled

to occasional bipolar cells, which are not their functional target (Trexler

et al. 2001). In other sites, such as cerebellum and thalamic reticular

nuclei, coupling is between the inhibitory neurons, but the principle cells

do not appear to express a connexin and thus are not ‘‘coupling compe-

tent’’. As well as inhibiting principle cells, these inhibitory interneurons

may form chemical inhibitory synapses with each other, and dendroden-

dritic inhibitory synapses can be found alongside dendrodendritic gap

junctions (Fukuda et al. 2006).Coupling of projection neurons is also well established at several sites.

Examples are the inferior olive, olfactory glomeruli (Rash et al. 2005),

locus coeruleus (Rash et al. 2007a), suprachiasmatic nuclei (Rash et al.

2007b), and various hypothalamic nuclei (see also reviews cited in preced-

ing paragraph). Electrical coupling of hippocampal pyramidal cells has

been reported (MacVicar and Ducek 1981, Mercer et al. 2006), although

there remains some controversy (Bennett and Pereda 2006). There is no

obvious incompatibility between hippocampal pyramidal cells that do and

do not form junctions with each other or between the inhibitory neurons

that make chemical synapses on them and also form gap junctions with

each other. Thus, neurons that express Cx36 do not necessarily form gap

junctions with each other, although they come in close enough contact for

chemical transmission.In mammals cortical pyramidal cells that project to the spinal cord can

form dual chemical and electrical synapses on spinal neurons (Rash et al.

2000), but do not couple in the cortex (Mercer et al. 2006). Conversely, in

the electromotor system of mormyrid fishes, medullary relay cells are

closely coupled by dendrodendritic gap junctions, but their axons transmit

chemically to the electromotor neurons in the spinal cord, which are

closely coupled to each other by dendrodendritic junctions (Bennett et al.

1967). In the electric eel, the medullary neurons are coupled to each other

and transmit electrically to the spinal electromotor neurons, which are

coupled by way of presynaptic fibers (as in Fig. 21.4C) and apparently

not by way of dendrodendritic connections (Meszler et al. 1974). Coupling

of postsynaptic neurons by way of presynaptic fibers in the absence of

direct dendrodendritic junctions may also occur in the vestibular nucleus


of the rat (Korn et al. 1973). In all of these examples regulatory mechan-isms in addition to connexin compatibility must be operative.

At most axodendritic and axosomatic synapses with gap junctions, thereare also structures suggestive of chemical transmission, and these apposi-tions are often called ‘‘mixed synapses.’’ Where the actual mode of transmis-sion has not been determined, a more precise characterization would be‘‘morphologically mixed.’’ Generally, mixed synapses in electromotor systemsof fishes transmit only electrically (Bennett 1972, 1977), and with weakstimulation of the VIIIth nerve forming the club endings on the Mauthnercell, there may be electrical transmission with little or no chemical compo-nent (Pereda et al. 2004). Stimulating a larger fraction of the fibers afferentto the Mauthner cell can increase the fraction of synapses that transmitchemically (apparently depolarization in the dendrite spreads antidromicallyinto the terminals and increases action potential amplitude), and tetanicstimulation can induce long-term potentiation of both components, evenwhen initially there is no chemical component in response to weak stimula-tion. Given the low open probability inferred for the gap junction channels,potentiation could result from change in channel open probability, ratherthan the insertion of new channels as suggested for glutamatergic synapses(Isaac 2003, Kerchner and Nicoll 2008). Since the fraction of open channelsat a gap junction can be quite small, the possibility should be consideredthat a morphologically described gap junction, whether or not at a mixedsynapse, may not have any open channels. However, no examples have beenreported to date.

21.7 Why Electrical Coupling?

Most of the classical properties of synapses can be observed at both chemi-cal and electrical synapses (Bennett 1972, 1977). An electrical connection isthought of as faster, and transmission starts without delay. However, ittakes time to charge the postsynaptic membrane capacity, and gap junctionsact as low-pass filters. The range of observed delays overlaps for the twoclasses of synapses and in mammals delay may be of little use in determin-ing the mode of transmission. With the possible exception of the vestibularnuclei of rat, a greater speed of transmission does not appear to be impor-tant for the function of gap junctions in the mammalian CNS (Korn et al.1973). There may be an advantage to electrical synapses, because theirreciprocity and linearity permit more effective synchronization of impulseactivity. Subthreshold as well as impulse signals can be transmitted, but thedegree of precision does not require the rapidity of electrical transmission.The reciprocal glutamatergic excitation between olfactory mitral cells doesdemonstrate the possibility of synchronization by chemical synapses (Chris-tie and Westbrook 2006, Pimentel and Margrie 2008).


21.8 Gap Junctions in Development

For some days after birth, most cortical neurons are dye coupled (Peinado et al.1993), probably via Cx45 junctions (Maxeiner et al. 2003). However, over timecoupling declines, and few if any pyramidal cells are coupled by day 14 (Yuste et al.1995). Synchronous firing appears to operate in the specification of neuronalcircuits (Yuste et al. 1995,Wong 1999, Butts et al. 2007).Metabolic communicationthrough gap junctions may also be important in establishing developmental gradi-ents of signaling molecues and allowing cells to choose different developmentalpaths.

21.9 A Fixation Artifact?

Although measurements of dye and electrical coupling are relatively straight-forward, artifacts are possible. For example, reports of dye coupling betweenfixed cells (Coleman and Sengelaub 2002) are suspect, because fixation isknown to block gap junctions in several tissues (Bennett 1973, Bushong et al.2002, Ogata and Kosaka 2002). However, fixation can promote cell fusion on amicroscopic scale that leads to dye and electrical coupling; cell fusion can beexcluded by application of a gap junction blocking agent or the injection oflarge molecular weight tracers, such as fluoresceinated dextrans or proteins(Bennett 1973). If cell fusion is produced by fixation, it implies that the cells areclosely apposed in vivo, probably without separation by astrocyte (glial)processes.

21.10 Pannexins/Innexins

Gap junctions in Cnidaria and protostomes are formed by a protein familyinitially termed innexins.When homologous genes were identified in the humangenome, the term pannexin was proposed for both vertebrate and invertebratehomologs (Shestopalov and Panchin 2008). This gene family is unrelated toconnexins (Yen and Saier 2007, Shestopalov and Panchin 2008). It can be statedwith confidence that connexins are absent in Drosophila, Caenorhabditis ele-gans, Nematostella (an anthozoan), and Hydra, since the genomes of thesespecies have been completely sequenced. Where in the deuterostome lineageconnexin genes first appeared has not been determined (see Putnam et al. 2007).Connexins are found in ascidians (Sasakura et al. 2003, White et al. 2004), andthe genome of Amphioxus has been sequenced, but the types of gap junctiongenes have not been reported (Putnam et al. 2008, Toyoda et al. 2008). Pannexins/innexins and connexins are stated to be absent in the genome of an echinoderm, thesea urchin Strongylocentrus purpuratus (Sea Urchin Genome Sequencing Consor-tium 2006). There are reports of electrical and dye coupling in echinoderms, so it is


likely that there are either analogs or homologs of the gap junction forming

proteins.Although not homologous with connexins, pannexins/innexins share many

structural and functional properties in common with them (Bruzzone et al.

2005, Scemes et al. 2007). Both classes are tetraspan proteins with cytoplasmic

N- and C-terminals. Both form hexameric hemichannels. Both have cysteine

groups in the two extracellular loops where formation of intramolecular dis-

ulphide bonds is likely required for structural stability. Both are sensitive to

similar blocking agents, including long chain alcohols (heptanol, octanol), high

intracellular Ca2+ and low intracellular pH. In both protein families, some

junctions are quite voltage insensitive to either or both transjunctional voltage

(Vj) and potential between the inside and outside of the cells, whereas others

show significant voltage dependence. Voltage dependence can be very fast,

probably due to rectification at the single channel level. Other junctions show

slower changes associated with changes in channel open probability, and there

can be distinct channel subconductance states. Both can open as hemichannels,

and in mammals, pannexins appear not to form cell–cell channels and to

function only as hemichannels in the surface membrane. This failure to form

gap junctions may be true in other vertebrates as well, although mammalian

pannexins will mediate electrical coupling when expressed in Xenopus oocytes.There are a few functional differences between pannexins/innexins and con-

nexins. Pannexin/innexin junctions are permeable to somewhat larger mole-

cules than connexin junctions (Simpson et al. 1977), and the intramembrane

particles seen in freeze fracture replicas are a bit larger. There is some indication

that, unlike connexin channels, pannexin/innexin channels can split in the plane

of the gap leaving hemichannels in each membrane that can then be reused

(Pappas et al. 1971, Lane and Swales 1978). Modern labeling techniques should

be applied to verify this inference. Innexins are in general encoded by multiple

exons.Most vertebrate connexins are encoded by a single exon; Cx36 is encoded

by two. Multiple exons encode connexins in ascidians. Identification of earlier

arising connexins may clarify whether introns have been lost or gained in

evolution.

References

Ahmad S and Evans WH (2002) Post-translational integration and oligomerization ofconnexin 26 in plasma membranes and evidence of formation of membrane pores:implications for the assembly of gap junctions. Biochem J 365:693–699

Altevogt BM and Paul DL (2004) Four classes of intercellular channels between glial cells inthe CNS. J Neurosci 24:4313–4323

Bartos M, Vida I and Jonas P (2007) Synaptic mechanisms of synchronized gamma oscilla-tions in inhibitory interneuron networks. Nat Rev Neurosci 8:45–56

Bennett MVL (1972) Electrical vs. chemical neurotransmission. In: Neurotransmitters. A.R.N.M.D 50:58–89


Bennett MVL (1973) Permeability and structure of electrotonic junctions and intercellularmovement of tracers. In: Kater SB andNicholson C (eds) Intracellular staining techniquesin neurobiology, Springer, New York, pp. 115–133

Bennett MVL (1977) Electrical transmission: a functional analysis and comparisonwith chemical transmission. In: Kandel ER (ed) Cellular biology of neurons Vol. I, Sec.I, Handbook of Physiology. The nervous system, Williams and Wilkins, Baltimore,pp. 357–416

Bennett MVL (2009) Gap junctions and electrical synapses. In: Squire LR (ed.) Encyclopediaof Neuroscience, vol. 4, pp. 529–548, Oxford, Academic Press

Bennett MVL, Contreras JE, Bukauskas FF et al. (2003) New roles for astrocytes: gapjunction hemichannels have something to communicate. Trends Neurosci 26:610–617

Bennett MV, Pappas GD, Aljure E et al. (1967) Physiology and ultrastructure of electrotonicjunctions. II. Spinal and medullary electromotor nuclei in mormyrid fish. J Neurophysiol30:180–208

Bennett MVL and Pereda A (2006) Pyramid power: principal cells of the hippocampus unite!Brain Cell Biol 35:5–11

Bennett MVL and Zukin RS (2004) Electrical coupling and neuronal synchronization in themammalian brain. Neuron 41:495–511

Bruzzone R, BarbeMT, JakobNJ et al. (2005) Pharmacological properties of homomeric andheteromeric pannexin hemichannels expressed in Xenopus oocytes. J Neurochem92:1033–1043

Bukauskas FF (2001) Inducing de novo formation of gap junction channels. Methods MolBiol 154:379–393

Bukauskas FF, Jordan K, Bukauskiene A et al. (2000) Clustering of connexin 43-enhancedgreen fluorescent protein gap junction channels and functional coupling in living cells.Proc Natl Acad Sci USA 97:2556–2561

Bukauskas FF, KreuzbergMM,RackauskasM et al. (2006) Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: implications for atrioventricular conduction inthe heart. Proc Natl Acad Sci USA 103:9726–9731

Bushong EA, Martone ME, Jones YZ et al. (2002) Protoplasmic astrocytes in CA1 stratumradiatum occupy separate anatomical domains. J Neurosci 22:183–192

Butts DA, Kanold PO and Shatz CJ (2007) A burst-based ‘‘Hebbian’’ learning rule at retino-geniculate synapses links retinal waves to activity-dependent refinement. PLoS Biol 5:e61

Christie JM and Westbrook GL (2006) Lateral excitation within the olfactory bulb.J Neurosci 26:2269–2277

Coleman AM and Sengelaub DR (2002) Patterns of dye coupling in lumbar motor nuclei ofthe rat. J Comp Neurol 454:34–41

Connors BW and Long MA (2004) Electrical synapses in the mammalian brain. Annu RevNeurosci 27:393–418

CotrinaML, Lin JH and NedergaardM (2008) Adhesive properties of connexin hemichannels.Glia 56:1791–1798

Cruikshank SJ, Landisman CE, Mancilla JG et al. (2005) Connexon connexions in thethalamocortical system. Prog Brain Res 149:41–57

Elias LA, Wang DD and Kriegstein AR (2007) Gap junction adhesion is necessary for radialmigration in the neocortex. Nature 448:901–907

Fukuda T, Kosaka T, Singer W et al. (2006) Gap junctions among dendrites of corticalGABAergic neurons establish a dense and widespread intercolumnar network. J Neurosci26:3434–3443

Fukuda T andKosaka T (2000) The dual network of GABAergic interneurons linked by bothchemical and electrical synapses: a possible infrastructure of the cerebral cortex. NeurosciRes 38:123–130

Gaietta G, Deerinck TJ, Adams SR et al. (2002)Multicolor and electron microscopic imagingof connexin trafficking. Science 296:503–507


Gibson JR, Beierlein M and Connors BW (1999) Two networks of electrically coupledinhibitory neurons in neocortex. Nature 402:75–79

Giepmans BN (2004) Gap junctions and connexin-interacting proteins. Cardiovasc Res62:233–245

Gumpert AM, Varco JS, Baker SM et al. (2008) Double-membrane gap junction inter-nalization requires the clathrin-mediated endocytic machinery. FEBS Lett582:2887–2892

Houades V, Koulakoff A, Ezan P et al. (2008) Gap junction-mediated astrocytic networks inthe mouse barrel cortex. J Neurosci 28:5207–5217

Isaac JT (2003) Postsynaptic silent synapses: evidence and mechanisms. Neuropharmacology45:450–460

Kerchner GA and Nicoll RA (2008) Silent synapses and the emergence of a postsynapticmechanism for LTP. Nat Rev Neurosci 9:813–825

Kleopa KA, Orthmann JL, Enriquez A et al. (2004) Unique distributions of the gapjunction proteins connexin29, connexin32, and connexin47 in oligodendrocytes. Glia47:346–357

Korn H, Sotelo C and Crepel F (1973) Electronic coupling between neurons in the rat lateralvestibular nucleus. Exp Brain Res 16:255–275

Koval M (2006) Pathways and control of connexin oligomerization. Trends Cell Biol16:159–166

KreuzbergMM,Deuchars J,Weiss E et al. (2008) Expression of connexin30.2 in interneuronsof the central nervous system in the mouse. Mol Cell Neurosci 37:119–134

Lane NJ and Swales LS (1978) Changes in the blood-brain barrier of the central nervoussystem in the blowfly during development, with special reference to the formation anddisaggregation of gap and tight junctions. Dev Biol 62:415–431

Li X, Ionescu AV, Lynn BD et al. (2004) Connexin47, connexin29 and connexin32co-expression in oligodendrocytes and Cx47 association with zonula occludens-1 (ZO-1)in mouse brain. Neuroscience 126:611–630

MacVicar BA and Dudek FE (1981) Electrotonic coupling between pyramidal cells: a directdemonstration in rat hippocampal slices. Science 213:782–785

Mann-Metzer P and Yarom Y. (2000) Electrotonic coupling synchronizes interneuron activityin the cerebellar cortex. Prog Brain Res 124:115–122

Maxeiner S, Kruger O, Schilling K et al. (2003) Spatiotemporal transcription of connexin45during brain development results in neuronal expression in adult mice. Neuroscience119:689–700

Mazet F,Wittenberg BA and Spray DC (1985) Fate of intercellular junctions in isolated adultrat cardiac cells. Circ Res 56:195–204

Maeda S,Nakagawa S, SugaM et al. (2009) Structure of the connexin 26 gap junction channelat 3.5 A resolution. Nature 458:597–602

Mege RM, Matsuzaki F, Gallin WJ et al. (1988) Construction of epithelioid sheets bytransfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules.Proc Natl Acad Sci USA 85:7274–7278

Mercer A, Bannister AP and Thomson AM (2006) Electrical coupling between pyramidalcells in adult cortical regions. Brain Cell Biol 35:13–27

Meszler RM, Pappas GD and Bennett VL (1974) Morphology of the electromotor system inthe spinal cord of the electric eel, Electrophorus electricus. J Neurocytol 3:251–261

Meyer RA, Laird DW, Revel JP et al. (1992) Inhibition of gap junction and adherens junctionassembly by connexin and A-CAM antibodies. J Cell Biol 119:179–189

Muller DJ, Hand GM, Engel A et al. (2002) Conformational changes in surface structures ofisolated connexin 26 gap junctions. EMBO J 21:3598–3607

Musil LS and Goodenough DA (1993) Multisubunit assembly of an integral plasma mem-brane channel protein, gap junction connexin43, occurs after exit from the ER. Cell74:1065–1077


Nagy JI, Dudek FE and Rash JE (2004) Update on connexins and gap junctions inneurons and glia in the mammalian nervous system. Brain Res Brain Res Rev47(1–3):191–215

Nagy JI, Ionescu AV, Lynn BD et al. (2003) Coupling of astrocyte connexins Cx26, Cx30,Cx43 to oligodendrocyte Cx29, Cx32, Cx47: implications from normal and connexin32knockout mice. Glia 44:205–218

NakajimaY (1974) Fine structure of the synaptic endings on theMauthner cell of the goldfish.J Comp Neurol 156:379–402

Ogata K and Kosaka T (2002) Structural and quantitative analysis of astrocytes in the mousehippocampus. Neuroscience 113:221–233

Oliveira R, Christov C, Guillamo JS et al. (2005) Contribution of gap junctional communica-tion between tumor cells and astroglia to the invasion of the brain parenchyma by humanglioblastomas. BMC Cell Biol 6:7

Orthmann-Murphy JL, Freidin M, Fischer E et al. (2007) Two distinct heterotypic channelsmediate gap junction coupling between astrocyte and oligodendrocyte connexins. J Neu-rosci 27:13949–13957

Pappas GD, Asada Y and Bennett MVL (1971) Morphological correlates of increasedcoupling resistance at an electrotonic synapse. J Cell Biol 49:173–188

PeinadoA, Yuste R andKatz LC (1993) Gap junctional communication and the developmentof local circuits in neocortex. Cereb Cortex 3:488–498

Pereda AE, Rash JE, Nagy JI et al. (2004) Dynamics of electrical transmission at club endingson the Mauthner cells. Brain Res Brain Res Rev 47:227–244

Pimentel DO and Margrie TW (2008) Glutamatergic transmission and plasticity betweenolfactory bulb mitral cells. J Physiol 586:2107–2119

Preus D, Johnson R, Sheridan J et al. (1981) Analysis of gap junctions and formationplaques between reaggregating Novikoff hepatoma cells. J Ultrastruct Res77:263–276

Prochnow N and Dermietzel R (2008) Connexons and cell adhesion: a romantic phase.Histochem Cell Biol 130:71–77

Prowse DM, Cadwallader GP and Pitts JD (1997) E-cadherin expression can alter thespecificity of gap junction formation. Cell Biol Int 21:833–843

PutnamNH, Butts T, Ferrier DE et al. (2008) The amphioxus genome and the evolution of thechordate karyotype. Nature 453:1064–1071

Putnam NH, Srivastava M, Hellsten U et al. (2007) Sea anemone genome reveals ancestraleumetazoan gene repertoire and genomic organization. Science 317:86–94

Rash JE, Staines WA, Yasumura T et al. (2000) Immunogold evidence that neuronal gapjunctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 orconnexin-43. Proc Natl Acad Sci USA 97:7573–7578

Rash JE, Davidson KG, Kamasawa N et al. (2005) Ultrastructural localization of connexins(Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa,olfactory nerve and olfactory bulb. J Neurocytol 34:307–341

Rash JE, Olson CO, DavidsonKG et al. (2007a) Identification of connexin36 in gap junctionsbetween neurons in rodent locus coeruleus. Neuroscience 147:938–956

Rash JE, Olson CO, Pouliot WA et al. (2007b) Connexin36 vs. connexin32, ‘‘miniature’’neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmaticnucleus. Neuroscience 149:350–371

Raviola E and Gilula NB (1973) Gap junctions between photoreceptor cells in the vertebrateretina. Proc Natl Acad Sci USA 70:1677–1681

Sasakura Y, Shoguchi E, Takatori N et al. (2003) A genomewide survey of developmentallyrelevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix.Dev Genes Evol 213:303–313

Scemes E, Suadicani SO, Dahl G et al. (2007) Connexin and pannexin mediated cell-cellcommunication. Neuron Glia Biol 3:199–208


Sea Urchin Genome Sequencing Consortium (2006) The genome of the sea urchin Strongy-locentrotus purpuratus. Science 314: 941–952

Segretain D and Falk MM (2004) Regulation of connexin biosynthesis, assembly, gap junc-tion formation, and removal. Biochim Biophys Acta 1662:3–21

Shaw RM, Fay AJ, Puthenveedu MA et al. (2007) Microtubule plus-end-tracking proteinstarget gap junctions directly from the cell interior to adherens junctions. Cell 128:547–560

Shestopalov VI and Panchin Y (2008) Pannexins and gap junction protein diversity. Cell MolLife Sci 65:376–394

Simpson I, Rose B and Loewenstein WR (1977) Size limit of molecules permeating thejunctional membrane channels. Science 195:294–296

Sohl G andWillecke K (2004) Gap junctions and the connexin protein family. Cardiovasc Res62:228–232

Thomas MA, Huang S, Cokoja A et al. (2002) Interaction of connexins with protein partnersin the control of channel turnover and gating. Biol Cell 94:445–456

Thomas T, JordanK and LairdDW (2001) Role of cytoskeletal elements in the recruitment ofCx43-GFP and Cx26-YFP into gap junctions. Cell Commun Adhes 8:231–236

Toyoda A, Bronner-Fraser M, Fujiyama A et al. (2008) The amphioxus genome and theevolution of the chordate karyotype Nature 453:1064–1071

Trexler EB, LiW,Mills SL et al. (2001) Coupling fromAII amacrine cells to ON cone bipolarcells is bidirectional. J Comp Neurol 437:408–422

Tuttle R,Masuko S andNakajimaY (1986) Freeze-fracture study of the largemyelinated clubending synapse on the goldfishMauthner cell: special reference to the quantitative analysisof gap junctions. J Comp Neurol 246:202–211

VanSlyke JK and Musil LS (2005) Cytosolic stress reduces degradation of connexin43 inter-nalized from the cell surface and enhances gap junction formation and function. Mol BiolCell 16:5247–5257

Wei CJ, Francis R, Xu X et al. (2005) Connexin43 associated with an N-cadherin-containingmultiprotein complex is required for gap junction formation in NIH3T3 cells. J Biol Chem280:19925–19936

White TW, Wang H, Mui R et al. (2004) Cloning and functional expression of invertebrateconnexins from Halocynthia pyriformis. FEBS Lett 577:42–48

WongRO (1999) Retinal waves and visual system development. AnnuRevNeurosci 22:29–47Yamane Y, Shiga H, Asou H et al. (1999) Dynamics of astrocyte adhesion as analyzed by a

combination of atomic force microscopy and immuno-cytochemistry: the involvementof actin filaments and connexin 43 in the early stage of adhesion. Arch Histol Cytol6:355–361

Yen MR and Saier MH Jr (2007) Gap junctional proteins of animals: the innexin/pannexinsuperfamily. Prog Biophys Mol Biol 94:5–14

Yuste R, Nelson DA, Rubin WW et al. (1995) Neuronal domains in developing neocortex:mechanisms of coactivation. Neuron 14:7–17

Zhang JT, Chen M, Foote CI et al. (1996) Membrane integration of in vitro-translated gapjunctional proteins: co- and post-translational mechanisms. Mol Biol Cell 7:471–482

Zampighi GA, Eskandari S and KremanM (2000) Epithelial organization of the mammalianlens. Exp Eye Res 71:415–435

Zhang JT, Chen M, Foote CI et al. (1996) Membrane integration of in vitro-translated gapjunctional proteins: co- and post-translational mechanisms. Mol Biol Cell 7:471–482
