gap junctional hemichannels in the heart

REVIEW

Gap junctional hemichannels in the heart

S. John, D. Cesario and J. N. Weiss

UCLA Cardiovascular Research Laboratory, Department of Medicine (Cardiology) and Physiology, The David Geffen School of Medicine

at UCLA, Los Angeles, CA, USA

Received 18 June 2003,

accepted 11 July 2003

Correspondence: Scott John,

UCLA Cardiovascular Research

Laboratory, 3645 MRL Bldg., The

David Geffen School of Medicine

at UCLA, Los Angeles, CA 90095-

1760, USA.

Abstract

Upon contacting each other, cells form gap junctions, in which each cell

contributes half of the channel linking their cytoplasms, enabling them to

share their metabolome up to a molecular weight of 1000. Each hemichannel

(or connexon) is randomly inserted into the plasma membrane and then

migrates to the site of cell-to-cell contact before pairing with the neigh-

bouring cell’s hemichannel to form a communicating conduit. This review

summarizes the evidence for hemichannels in heart ventricular myocytes.

Morphological findings are summarized describing how hemichannels are

inserted into the plasma membrane. Once in the plasma membrane, hemi-

channels can be functionally detected electrophysiologically or by dye uptake

assays. Each technique reveals specific aspects of hemichannel function.

Using dye uptake studies, it is possible to investigate the biological regulation

of hemichannels in vivo. Evidence is summarized which indicates that

hemichannels are normally kept closed in the presence of normal extracel-

lular Ca because they are phosphorylated at residues in the C-terminus

regulated by the MAPK signalling pathway. When hemichannels are

dephosphorylated, the channels open and allow dye uptake into the cells, as

well as potentially deleterious ion exchange. Biological stresses, such as

hyperosmolarity and metabolic inhibition, open hemichannels by this

mechanism through activating phosphatases. The resulting ion fluxes may

have important roles in heart physiology and pathophysiology.

Keywords connexin, connexons, gap junctions, hemichannels.

Cell-to-cell communication depends on both extracel-

lular and intracellular pathways. The extracellular

pathway uses hormones and neurotransmitters to

transmit information. The intracellular pathway is

mediated by specialized plasma membrane structures

called gap junctions. Gap junctions are bipartite struc-

tures, each cell contributing half of the working gap

junction. This inevitably means that ‘half-gap junctions’

must exist at least immediately prior to the formation of

the gap junction. Half-gap junctions have been called

hemichannels or connexons. These structures are

formed by the oligomerization of transmembrane pro-

teins called connexins, six of which form an individual

connexon. A multigene family encodes the connexin

proteins and more than 20 connexins have now been

identified.

Most research on connexins has focused on how they

form and function as gap junctions. Hemichannels have

been studied as a reductionist means to characterize gap

junctions, and in this context have been identified in

many cell types using an array of biochemical and

electrophysiological techniques. However, there is a

growing body of evidence suggesting that the hemi-

channels have additional functions beyond their role as

precursors to gap junction channels. Functionality of

hemichannels has been documented by showing that

they allow dyes of low molecular weight to enter the cell

from the extracellular environment, or induce non-

Acta Physiol Scand 2003, 179, 23–31

� 2003 Scandinavian Physiological Society 23

selective currents under some conditions. Recently,

studies have addressed whether these properties have

any biological roles under physiological and pathophys-

iological conditions.

Morphological features of hemichannels

Gap junctions are detected by their characteristic

morphological appearance in thin section negative stain

electron microscopy (EM) images (Revel & Karnovsky

1967). EM, particularly freeze fracture, shows para-

crystalline arrays of intramembranous particles com-

prising a plaque. A plaque can range in size from just a

few particles to as many as 200 000 particles. Freeze-

fracture studies have also shown that plaques grow by

accretion (Ryerse et al. 1984). A small plaque forms,

around which there is a particle free zone. Outside of

this zone are more intramembranous particles, which

are the same size and have the same freeze fracture

characteristics as those within the plaque. Similar

results have been elegantly obtained using real time

fluorescence microscopy to monitor gap junction for-

mation (Gaietta et al. 2002). Connexin43, the most

predominant connexin of the three connexins found in

the heart, was tagged with a tetra-cysteine repeat amino

acid sequence, which enabled membrane permeant dyes

to bind. These dyes, known as bioarsenical fluoroph-

ores, come in two different colours, green and red. After

labelling with the green fluorophore, bright green

fluorescent lines appeared between contacting cells,

which were shown to be gap junctions by EM section-

ing. Following a time delay of several hours and

washout of the green dye, the red fluorophore was

applied to label only newly synthesized connexin

protein. The interfacial regions of cells now showed

green lines bounded by red (see Fig. 1). These results

demonstrate that gap junctions grow by accretion of

connexons. Thus, hemichannels are initially inserted

into the plasma membrane before they migrate to the

gap junction. These results are similar to the findings of

the earliest freeze fracture studies of gap junctions.

Morphological evidence for hemichannels has come

from immunocytochemical studies. A few studies have

shown the presence of Cx43 in non-contacting regions of

neonatal heart cells, i.e. in non-gap-junctional regions (el

Aoumari et al. 1991, Rook et al. 1992). More recent

studies have shown that when connexin43 is linked to the

green fluorescent protein (GFP), connexin protein can be

found in non-contacting regions of the cell membrane of

transfected mammalian cells (John et al. 1999) (Figs 1

and 2). Poor resolution at the light microscope level

makes it difficult to determine whether these connexins

represent closely associated collections of single protein

molecules, individual subunits, or packed subunits.

However, plaques of hemichannels have been

observed. Using the atomic force microscope (AFM),

Figure 1 Connexin43 insertion into the plasma membrane. Photoconversion of DAB by ReAsH enables electron microscopy of

connexin43 tagged sequentially with ReAsh-EDT2 (green) followed by ReAsH (red) to label newly synthesized connexin43 into

both gap junctional plaques and the plasma membrane. The green comes from ReAsh-EDT2, which under particular experimental

conditions only labels newly synthesized connexin 43. (a) Confocal image of a labelled junctional plaque. (b) Correlated EM

image after photoconversion, showing staining of the peripheral portions of the junction and newly created gap junctions (arrows).

(d)–(f) show staining of vesicles (arrows) associated with the Golgi apparatus (d), putative transport vesicles (e), and an exocytic

event (f). The last image details the observation of connexin43 being inserted into the membrane in a region of the cell absent

contact with another cell. Bars in (a) and (b) 2 lm and in (d)–(f) 0.5 lm. Reprinted with permission, from Gaietta et al. (2002).

24 � 2003 Scandinavian Physiological Society

Gap junctional hemichannels in the heart Æ S John et al. Acta Physiol Scand 2003, 179, 23–31

with preparations originally used to isolate heart gap

junctions, Lal et al. (1995) visualized plaques which

had characteristics similar to native heart gap junctions

except for their thickness of 9–11 nm, as compared to

22–27 nm for gap junctions (Fig. 3). These hemichannel

plaques showed long-range hexagonal packing with

similar centre-to-centre spacing (9–10 nm), and decrease

in thickness to 6–9 nm when treated with trypsin, which

Figure 3 Atomic Force Microscopy (AFM) images of hemiplaques isolated from rat heart. Images were obtained in a buffered

solution containing 30% dextrose. (a) Low resolution surface view of a presumptive isolated gap junctional hemiplaque. The scan

dimensions are 0.9 lm · 0.9 lm. The measured thickness of the membrane is �9.6 nm, about half the height of an intact cardiac

gap junction plaque. (b) Inverse Fourier transformed image showing good long range packing of connexons, with a centre-to-centre

spacing of 9.7 nm. (c) Increased resolution image showing subunits on the surface of the hemiplaque. The scan dimensions are

130 nm · 130 nm. (d) High resolution image of an individual connexon. The central pore (�2.5 nm in diameter) of the connexon is

visible with its surrounding subunits. Scan size is 10 nm · 10 nm. Reprinted with permission, from Lal et al. (1995).

Figure 2 Cellular distribution of GFP and Cx43 fused to GFP (Cx43GFP) in HEK293 cells. (a) Brightfield image of HEK293

cells transfected with a plasmid encoding GFP. (b) Fluorescent image of the cell expressing GFP filling the cytoplasm of

the transfected cell. (c and d) Brightfield and fluorescent images of cells expressing Cx43GFP. Note the prominent fluorescent

line between the cells consistent with the formation of gap junctions, similar to that observed in Figure 1. Fluorescent labelling

of intracellular regions and regions at or near the plasma membrane indicate the biosynthetic pathway of connexin43 and may

show incorporation into regions of the plasma membrane not in contact with other cells, i.e. hemichannels. Reprinted with

permission, from John et al. (1999).


Acta Physiol Scand 2003, 179, 23–31 S John et al. Æ Gap junctional hemichannels in the heart

has been shown to remove the cytoplasmic tail from

Cx43. More recent AFM studies have shown that

connexin 26 hemichannels respond to changes in extra-

cellular Ca concentration. Using the AFM as a nanoma-

nipulator, Muller et al. (2002) removed one half of the

gap junction layer to reveal the extracellular face of the

apposing hemichannel. They showed that the extracel-

lular pore dimensions decreased from 1.6 to 0.6 nm

when Ca concentrations were increased from 0 to

0.5 mm (Fig. 4). The permeability changes that this

change in pore size caused were not investigated, but it

suggests that Ca on the extracellular face is capable of

directly gating the channels.

Functional assessment of hemichannels

To characterize hemichannel function, various criteria

must be met. Using electrophysiological approaches, the

hemichannel must conduct ions almost non-selectively,

typically with a reversal potential near zero (Fig. 5). Using

dye permeation, the channel must allow passage of dyes of

up to �1000 MW, but exclude larger dyes (>1500) (Fig.

6). The charge of the dye may affect its permeation

depending on the connexin composition of the hemichan-

nel, i.e. connexons show some selectivity, usually for

cations over anions. Gap junction blockers should block

(usually only partially) the non-selective current through

hemichannels, and inhibit permeation of dyes <1000 MW.

In addition, removal of extracellular Ca2+ should open the

permeation pathway. For molecular biology studies, it is

ideal to express connexins in cells, which either lack

functional gap junctions, or do not express connexin genes.

In this case, overexpression of exogenous connexins

should generate a new conductance and dye permeability

defined by the criteria outlined above.

Hemichannels have been characterized in a variety

of systems, including reconstitution in lipid bilayers,

heterologous expression of connexin proteins in

Xenopus oocytes or mammalian cell lines lacking

endogenous connexins, endogenous hemichannels in

Figure 4 Extracellular surface view of Cx26 connexons, in the absence of Ca (a) and upon addition of Ca (b) (0.5 mm Ca).

Correlation averages are shown taken from several topographs recorded under equivalent conditions. The lateral resolution of

the images was limited to �1.5 nm. As the Ca concentration was increased the central pore diameter decreased from 1.6 nm in

the absence of Ca to 0.6 nm in the presence of 0.5 mm Ca. Reprinted with permission, from Muller et al. (2002).

Figure 5 Activation of hemichannel currents by low extra-

cellular Ca in HEK293 cells expressing Cx43-GFP: (a) time

course activation of whole cell hemichannel currents by low

extracellular Ca (0Ca) in a patch-clamped HEK293 cell

transfected with Cx43-GFP. Current amplitude was measured

at )80 mV. The current was reversibly blocked by the appli-

cation of halothane (H). (b) Current–voltage relationships at

the selected time points (a–d) indicated in (a). Currents were

recorded during a voltage ramp from )100 to +60 mV at

0.1 mV ms)1. Selected time points were as follows: a, control

with 1.8 m [Ca]o; b, after the removal of extracellular Ca

(0Ca); c, after the reapplication of 1.8 mm [Ca]o; d, in the

presence of halothane (H) after removing extracellular Ca

again. Non-transfected cells never showed this response.

Reprinted with permission, from John et al. (1999).



primary cell cultures, Novikoff Hepatoma cells (Li

et al. 1996), astrocytes (Hofer & Dermietzel 1998),

and some native tissues that express hemichannels such

as horizontal cells from the retina of catfish or skate

(DeVries & Schwartz 1992, Malchow et al. 1993),

astrocytes (Hofer & Dermietzel 1998) and ventricular

myocytes (John et al. 1999, Kondo et al. 2000).

Biological regulation of hemichannels

We became interested in cardiac hemichannels because

of their potential role in response to osmotic challenges

and metabolic inhibition in the heart. Here, we

summarize some of the background evidence for their

involvement in these processes in non-cardiac tissues.

Using AFM to monitor volume changes, Quist et al.

(2000) showed that cells lacking endogenous connexins

failed to regulate their volume appropriately in response

to extracellular Ca changes, unless they were transfect-

ed with Cx43. The gap junction blockers oleamide and

18-a-glycyrrhetinic acid also inhibited the response in

Cx43 transfected cells. Indirect evidence for a role of

hemichannels in volume regulation was provided by

Paul et al. (1991), who found that when Cx46 hemi-

channels were expressed in Xenopus oocytes, the

oocytes died unless Ficoll was included in the extracel-

lular buffer to maintain osmotic balance. Later studies

showed that the Cx46 open hemichannels could be

Figure 6 Dye uptake and transfer in HEK293 cells. (a) and (b) Double exposure of bright-field and fluorescence images (a) or

the fluorescence image alone (b) of a monolayer of non-transfected HEK293 cells after exposure to 0Ca solution containing 1%

Lucifer Yellow for 30 min. Occasionally, dead cells took up Lucifer Yellow (inset) but did not transfer the dye to adjacent cells. (c)

and (d) double exposure of bright-field and fluorescence images (e) and the fluorescence image alone (f) of a monolayer of HEK293

cells transfected with Cx43-GFP after exposure to 0Ca solution containing Lucifer Yellow for 30 min. The fluorescence from Cx43-

GFP is very bright and localized at discrete spots, as seen in Figure 1. Lucifer Yellow fluorescence is most intense in the Cx43-

GFP-labelled cells and becomes dimmer in the adjacent non-transfected cells, which is consistent with dye transfer. In contrast,

the more distant non-transfected cells did not show any Lucifer Yellow fluorescence. (e) and (f) Double exposure of bright-field

and fluorescence images (c) and the fluorescence image alone (d) of a monolayer of HEK293 cells transfected with wild-type Cx43

after exposure to 0Ca solution containing Lucifer Yellow for 30 min. Note the bright fluorescence of the (presumably) transfected

cell in the upper left corner, with dimmer fluorescence consistent with dye transfer to surrounding cells. In contrast, distant cells

show no fluorescence. Reprinted with permission, from John et al. (1999).



closed by raising extracellular Ca levels (Ebihara &

Steiner 1993).

When neuronal astrocytes were treated with chemi-

cals to mimic ischaemia, such as inhibitors of glycolytic

and oxidative metabolism, hemichannels opened, as

judged by the permeability to the low molecular weight

dyes Lucifer Yellow and ethidium bromide (Contreras

et al. 2002). The non-specific gap junctional inhibitors,

octanol and 18-a-glycyrrhetinic acid, decreased the

cellular uptake of these dyes. In studies of astrocytes

obtained from mice deficient for Cx43, no dye uptake

was observed when subjected to metabolic inhibition

(Contreras et al. 2002). The response of hemichannels

to metabolic inhibition was linked to the phosphoryla-

tion state of Cx43. During metabolic inhibition, levels

of dephosphorylated Cx43 were shown to increase

coincident with ethidium bromide dye uptake. How-

ever, as dephosphorylation was not complete, it was not

resolved conclusively whether hemichannels were

opened by dephosphorylation. Others have shown using

cultured astrocytes that hypoxia-induced Cx43 dep-

hosphorylation was mediated by calcineurin (Li et al.

1998); however, gap junctional communication rather

than hemichannel activity was being examined.

Direct evidence that hemichannel function is regula-

ted by phosphorylation was reported by Kim et al.

(1999), who showed that when Cx43 was incorporated

into unilamellar vesicles, a fraction exhibiting sucrose

permeability was also permeable to Lucifer Yellow,

consistent with functional hemichannels. Treating the

Cx43 with calf alkaline phosphatase either before or

after reconstitution in the vesicles increased the vesicle

permeability. Moreover, MAP kinase treatment to

phosphorylate Cx43 decreased the permeability of the

vesicles. Warn-Cramer et al. (1996) had previously

identified residues on Cx43 which are phosphorylated

by MAP kinase (S255 S279 and S282), although these

studies were performed in gap junction channels rather

than hemichannels. Regulation of Cx43 gap junctions

by other kinases such as protein kinase A and C have

also been demonstrated (for review see, Goodenough

et al. 1996).

Hemichannels in heart

Three major connexins are expressed in the heart,

Cx40, Cx43 and Cx45 (Coppen et al. 1999a). Cx40

and Cx45 are found predominantly in the conduction

system (Coppen et al. 1999b), whereas Cx43 is ubi-

quitous. Most studies of connexins in heart have

focused on their role in gap junctions, but some studies

have characterized Cx43 hemichannels in heart cells.

John et al. 1999 and Kondo et al. 2000 examined

isolated adult rabbit ventricular myocytes to see if

functional hemichannels could be detected. Either

removal of extracellular Ca, or metabolic inhibition in

the presence of normal extracellular Ca, induced a dye

permeable pathway, which allowed the entry of the low

molecular weight dye Calcein (625 MW), but excluded

the high molecular weight dye dextran rhodamine

(3000 MW) (Fig. 7). Kondo et al. (2000) characterized

this pathway electrophysiologically during extracellular

Ca removal and metabolic inhibition, and found a non-

selective current was activated by these interventions

(Fig. 8). These results suggested that hemichannels from

adult cardiac myocytes exist in vivo. It is unlikely that

these hemichannels were an artefact of the cell isolation

procedure, as EM studies have shown that gap junctions

are pulled off cells as membrane vesicles, rather than

splitting apart to form arrays of hemichannels (Severs

1995). The definitive proof of the existence of hemi-

channels in the heart, however, still requires their

(a) (b)

Figure 7 Size-selective dye uptake by isolated rabbit ventricular myocytes exposed to Ca-free media. (a) and (b) Representative

paired bright-field and fluorescence images of isolated myocytes after a 30-min incubation with either (a) 150 mm calcein (Mr 660)

or (b) 1% dextran conjugated to fluorescein (Mr 1500–3000) in the presence (1Ca) or absence (0Ca) of 1.8 mm Ca. Note that live

rod-shaped cells take up calcein only in the absence of Ca, whereas rounded dead cells take up both dyes irrespective of Ca.

Reprinted with permission, from John et al. (1999).



demonstration in intact tissue. This has not yet been

achieved, but preliminary results from our laboratory

indicate that size-selective dye uptake occurs during

extracellular Ca removal or hypoxia in the isolated

arterially perused rabbit ventricular septum, supporting

the conjecture of functioning hemichannels in intact

cardiac muscle.

Consistent with the findings in isolated ventricular

myocytes, John et al. (1999) also demonstrated that

when Cx43 was expressed in non-confluent HEK293

cells (either as wildtype Cx43 or fused to green fluores-

cent protein, GFP), functional hemichannels could be

demonstrated (Fig. 6). They characterized hemichannel

functionality by incubating Cx43 transfected cells in the

presence or absence of extracellular Ca. Upon removal of

extracellular Ca, a permeant pathway opened which

allowed for the extracellular dye Lucifer Yellow to enter

the cells. When patched in the whole cell patch clamp

mode, currents induced by extracellular Ca removal or

metabolic inhibition showed a reversal potential near

zero and were inhibited partially by octanol or the

lanthanum. The I–V curve from HEK cells showed little

voltage dependence over the range )100 to +60 mV (Fig.

5). These results were consistent with those of Li et al.

(1996), who showed that hemichannels expressed in

tissue culture cells and kidney cells were almost exclu-

sively Cadependent and insensitive to other divalent

cations. Unitary currents from hemichannels in cell-

attached patches were also detected by John et al. (1999)

with no Ca in the patch pipet, and during metabolic

inhibition, with a unitary conductance �120 pS.

Recent work from our laboratory has been focusing

on the mechanisms by which Cx43 hemichannels are

regulated. Initial studies compared the response of Cx26,

Cx31 and Cx43 to metabolic inhibition in the presence

of normal extracellular Ca. HeLa cells expressing either

Cx43 or Cx31 showed dye uptake consistent with

hemichannel opening. The gap junction blockers olei-

mide or 18-a-glycyrhetinic acid blunted the dye uptake

by 70 and 53%, respectively. We also found that in the

presence of normal extracellular Ca, hemichannels

opened in response to inhibiting PKC, PTK or MAPK

signalling (Cesario et al. 2002), with MAPK signalling

being the farthest downstream. Interestingly, these same

protein kinase signalling pathways are known to confer

cardioprotection during ischaemia. In subsequent stud-

ies, we have identified the serine residues at 255, 279 and

282 in the C-terminus of Cx43 as the sites responsible for

gating hemichannels by MAPK signalling, consistent

with the findings of Warn-Cramer et al. (1996).

Physiological significance of hemichannel

opening in the heart

Given that MAPK signalling is important in cellular

stress, such as responses to osmotic changes (Feranchak

et al. 2001), we investigated the effects of osmotic

stress on isolated ventricular myocytes and Hela cells

expressing Cx43. We found that, similar to metabolic

inhibition, as little as a 25 mOsm hyperosmotic chal-

lenge was sufficient to induce hemichannel opening, as

measured by size selective dye uptake, in both the Hela

and myocyte cells, and was associated with Cx43

dephosphorylation. The mechanism appeared to involve

phosphatase activation leading to Cx43 dephosphory-

lation, as MAPK activity was either unchanged or

increased by mild hyperosmotic stress. In addition,

Figure 8 Activation of hemichannel currents in isolated rabbit

ventricular myocytes by metabolic inhibition (a–c). (a) time

course of whole cell current at )80 mV in a patch-clamped

myocyte during MI with rotenone in the presence of 1.8 mm

[Ca]o, with all conventional ionic currents blocked by appro-

priate ionic substitutions and drugs. The current was blocked

by 2 mm lanthanum (La). (b) Current–voltage relationships at

the selected time points (a–c) indicated in (a). (c) Summary of

the effects of metabolic inhibition. Reprinted with permission,

from John et al. (1999).



okadaic acid prevented hemichannel opening by mild

hyperosmotic stress.

How might this osmoregulatory role of hemichan-

nels be important in the clinical setting? During

myocardial ischaemia, dramatic osmotic shifts occur

due to the accumulation of metabolic by-products such

as lactate and Pi, which may possibly cause hemichan-

nels to open. There are an estimated 2.6 million

connexons in each cardiac myocyte, the vast majority

linked to connexons from neighbouring myocytes in

gap junction plaques. Given a turnover rate of

90–120 min (Beardslee et al. 1998), if the time interval

between insertion in the sarcolemma and partnering

with a neighbouring connexon took 1 min, then at any

given moment there would be about �11 000 unpart-

nered connexons, i.e. hemichannels, in each myocyte.

From their measured single channel conductance of

�120 pS and typical open probability in zero Ca of

0.3, it can be estimated that opening of only 50

hemichannels would double the Na influx of a normal

beating myocyte (John et al. 1999). Thus, the ability of

the Na–K pump to compensate for the intracellular Na

gain and K loss would be swamped if even a small

percentage of the total number of hemichannels were

to open during metabolic inhibition. Furthermore, it is

known that Cx43 is dephosphorylated during myocar-

dial ischaemia (Beardslee et al. 2000), although whe-

ther this involves unpartnered connexons to the same

extent as partnered connexons is unknown. During

myocardial ischaemia, the activity of many protein

kinase pathways increases, but so does phosphatase

activity (Armstrong and Ganote 1992, Weinbrenner

et al. 1998). From direct measurements of Cx43

phosphorylation levels during ischaemia, however, the

balance of these factors, however, favours dephosph-

orylation (Jeyaraman et al. 2003). In our hypothetical

scenario, opening of dephosphorylated hemichannels

will contribute to the intracellular Na loading and K

loss during acute ischaemia. Thus, the rate of hemi-

channel dephosphorylation during ischaemia could be

an important determinant of susceptibility to isch-

aemia/reperfusion injury and ischemic arrhythmias, as

intracellular Na gain promotes intracellular Ca over-

load leading to reperfusion injury, and extracellular K

loss causes electrophysiological alterations often pro-

moting lethal cardiac arrhythmias. The different roles

of hemichannels and gap junctions may be important

under these conditions as open hemichannels allow Ca

overload, which would tend to close gap junctional

channels; thus, acting to limit the size of the infarct. In

addition, we speculate that by inducing a high level of

hemichannel phosphorylation immediately before the

onset of prolonged ischaemia, protein kinase C and

other protein kinase activators may be cardioprotective

by slowing the rate at which dephosphorylated

hemichannels open and cause deleterious ionic shifts.

Hemichannels may, therefore, also be important in

ischaemic preconditioning, as the preconditioning with

episodes of brief ischaemia also activates these same

protein kinase pathways.

This works was supported by NIH Score in Sudden Cardiac

Death P50 HL52319, and by the Kawata and Laubisch

Endowments.

References

Armstrong, S.C. & Ganote, C. E. 1992. Effects of the protein

phosphatase inhibitors okadaic acid and calyculin A on

metabolically inhibited and ischaemic isolated myocytes.

J Mol Cell Cardiol 24, 869–884.

Beardslee, M.A., Laing, J.G., Beyer, E.C. & Saffitz, J.E. 1998.

Rapid turnover of connexin43 in the adult rat heart. Circ Res

83, 629–635.

Beardslee, M.A., Lerner, D.L., Tadros, P.N. et al. 2000. Dep-

hosphorylation and intracellular redistribution of ventricular

connexin43 during electrical uncoupling induced by ische-

mia. Circ Res 87, 656–662.

Cesario, D., John, S.A. & Weiss, J.N. 2002. Hemichannels

opened by metabolic inhibition and protein kinase inhibi-

tion. Biophys J 82, 221a.

Contreras, J.E., Sanchez, H.A., Eugenin, E.A. et al. 2002.

Metabolic inhibition induces opening of unopposed conn-

exin 43 gap junction hemichannels and reduces gap junc-

tional communication in cortical astrocytes in culture. Proc

Natl Acad Sci USA 99, 495–500.

Coppen, S.R., Kodama, I., Boyett, M.R. et al. 1999a. Conn-

exin45, a major connexin of the rabbit sinoatrial node, is

co-expressed with connexin43 in a restricted zone at the

nodal-crista terminalis border. J Histochem Cytochem 47,

907–918.

Coppen, S.R., Severs, N.J. & Gourdie, R.G. 1999b. Con-

nexin45 (alpha 6) expression delineates an extended con-

duction system in the embryonic and mature rodent heart.

Dev Genet 24, 82–90.

DeVries, S.H. & Schwartz, E.A. 1992. Hemi-gap-junction

channels in solitary horizontal cells of the catfish retina.

J Physiol 445, 201–230.

Ebihara, L. & Steiner, E. 1993. Properties of a nonjunctional

current expressed from a rat connexin46 cDNA in Xenopus

oocytes. J Gen Physiol 102, 59–74.

el Aoumari, A., Dupont, E., Fromaget, C. et al. 1991. Immu-

nolocalization of an extracellular domain of connexin43 in

rat heart gap junctions. Eur J Cell Biol 56, 391–400.

Feranchak, A.P., Berl, T., Capasso, J., Wojtaszek, P.A., Han, J.

& Fitz, J.G. 2001. p38 MAP kinase modulates liver cell

volume through inhibition of membrane Na+ permeability.

J Clin Invest 108, 1495–1504.

Gaietta, G., Deerinck, T.J., Adams, S.R. et al. 2002. Multi-

color and electron microscopic imaging of connexin traf-

ficking. Science 296, 503–507.

Goodenough, D.A., Goliger, J.A. & Paul, D.L. 1996. Con-

nexins, connexons, and intercellular communication. Annu

Rev Biochem 65, 475–502.



Hofer, A. & Dermietzel, R. 1998. Visualization and functional

blocking of gap junction hemichannels (connexons) with

antibodies against external loop domains in astrocytes. Glia

24, 141–154.

Jeyaraman, M., Tanguy, S., Fandrich, R.R., Lukas, A. &

Kardami, E. 2003. Ischemia-induced dephosphorylation of

cardiomyocyte connexin-43 is reduced by okadaic acid and

calyculin A but not fostriecin. Mol Cell Biochem 242, 129–

134.

John, S.A., Kondo, R., Wang, S.Y., Goldhaber, J.I. & Weiss,

J.N. 1999. Connexin-43 hemichannels opened by metabolic

inhibition. J Biol Chem 274, 236–240.

Kim, D.Y., Kam, Y., Koo, S.K. & Joe, C.O. 1999. Gating

connexin 43 channels reconstituted in lipid vesicles by

mitogen-activated protein kinase phosphorylation. J Biol

Chem 274, 5581–5587.

Kondo, R.P., Wang, S.Y., John, S.A., Weiss, J.N. & Gold-

haber, J.I. 2000. Metabolic inhibition activates a non-selec-

tive current through connexin hemichannels in isolated

ventricular myocytes. J Mol Cell Cardiol 32, 1859–1872.

Lal, R., John, S.A., Laird, D.W. & Arnsdorf, M.F. 1995. Heart

gap junction preparations reveal hemiplaques by atomic

force microscopy. Am J Physiol 268, C968–C977.

Li, H., Liu, T.F., Lazrak, A. et al. 1996. Properties and regu-

lation of gap junctional hemichannels in the plasma mem-

branes of cultured cells. J Cell Biol 134, 1019–1030.

Li, W.E., Ochalski, P.A., Hertzberg, E.L. & Nagy, J.I. 1998.

Immunorecognition, ultrastructure and phosphorylation

status of astrocytic gap junctions and connexin43 in rat

brain after cerebral focal ischaemia. Eur J Neurosci 10,

2444–2463.

Malchow, R.P., Qian, H. & Ripps, H. 1993. Evidence for

hemi-gap junctional channels in isolated horizontal cells of

the skate retina. J Neurosci Res 35, 237–245.

Muller, D.J., Hand, G.M., Engel, A. & Sosinsky, G.E. 2002.

Conformational changes in surface structures of isolated

connexin 26 gap junctions. EMBO J 21, 3598–3607.

Paul, D.L., Ebihara, L., Takemoto, L.J., Swenson, K.I. &

Goodenough, D.A. 1991. Connexin46, a novel lens gap

junction protein, induces voltage-gated currents in non-

junctional plasma membrane of Xenopus oocytes. J Cell Biol

115, 1077–1089.

Quist, A.P., Rhee, S.K., Lin, H. & Lal, R. 2000. Physiological

role of gap-junctional hemichannels. Extracellular calcium-

dependent isosmotic volume regulation. J Cell Biol 148,

1063–1074.

Revel, J.P. & Karnovsky, M.J. 1967. Hexagonal array of

subunits in intercellular junctions of the mouse heart and

liver. J Cell Biol 33, C7–C12.

Rook, M.B., van Ginneken, A.C., de Jonge, B., el Aoumari, A.,

Gros, D. & Jongsma, H.J. 1992. Differences in gap junction

channels between cardiac myocytes, fibroblasts, and het-

erologous pairs. Am J Physiol 263, C959–C977.

Ryerse, J.S., Nagel, B.A. & Hammel, I. 1984. The role of

connexon aggregate fusion in gap junction growth. J Sub-

microsc Cytol 16, 649–657.

Severs, N.J. 1995. Microscopy of the gap junction: a historical

perspective. Microsc Res Tech 31, 338–346.

Warn-Cramer, B.J., Lampe, P.D., Kurata, W.E. et al. 1996.

Characterization of the mitogen-activated protein kinase

phosphorylation sites on the connexin-43 gap junction pro-

tein. J Biol Chem 271, 3779–3786.

Weinbrenner, C., Baines, C.P., Liu, G.S. et al. 1998. Fostriecin,

an inhibitor of protein phosphatase 2A, limits myocardial

infarct size even when administered after onset of ischemia.

Circulation 98, 899–905.



gap junctional hemichannels in the heart

Documents