Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Following tracks of hemichannels

It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled by means of antibodies directed to an external loop domain of connexin (Cx) 43. We now provide evidence that trafficking of hemichannel vesicles can be visualized upon binding of a labeled homophilic peptide corresponding to a region of the first extracellular loop (EL1) of Cx43. In vivo imaging was performed after labeling hemichannels from the extracellular site with a mimetic peptide tagged with a fluorochrome (Alexa-546). Using a Cx43-CFP transfected HeLa cell line for incubation with the mimetic peptide, a significant number of double-labeled vesicles were found inside the cells. This double labeling indicates that a portion of Cx43 within the cell had accessed the cell surface as hemichannels where it bound to the peptide and was subsequently endocytosed. Pulse labeling with the peptide showed a decrease in the number of dual-labeled vesicles over time, indicating degradation and/or concurrent recycling of hemichannel vesicles.     

Following Tracks of Hemichannels

It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled by means of antibodies directed to an external loop domain of connexin (Cx) 43 (1). We now provide evidence that trafficking of hemichannel vesicles can be visualized upon binding of a labeled homophilic peptide corresponding to a region of the first extracellular loop (EL1) of Cx43. In vivoimaging was performed after labeling hemichannels from the extracellular site with a mimetic peptide tagged with a fluorochrome (Alexa-546). Using a Cx43-CFP transfected HeLa cell line for incubation with the mimetic peptide, a significant number of double-labeled vesicles were found inside the cells. This double labeling indicates that a portion of Cx43 within the cell had accessed the cell surface as hemichannels where it bound to the peptide and was subsequently endocytosed. Pulse labeling with the peptide showed a decrease in the number of dual-labeled vesicles over time, indicating degradation and/or concurrent recycling of hemichannel vesicles.     

Manipulating Connexin Communication Channels: Use of Peptidomimetics and the Translational Outputs

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell’s plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.     

Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7- hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.     

Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium

The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor α-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation.     

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.     

Connexin channels, connexin mimetic peptides and ATP release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 mum from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.     

Direct Regulation of Osteocytic Connexin 43 Hemichannels through AKT Kinase Activated by Mechanical Stimulation

Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.     

Connexin Channels, Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃ elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Connexin Channels,Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.     

Connexin-43 hemichannels contribute to the propagation of μ-calpain-mediated neuronal death in a cortical ablation injury model

We investigated the role of the astrocytic and neuronal hemichannels (HCs) in the spread of cortical neuronal death in a rat cortical injury model. Over time (by 6 h), propidium iodide (PI)-positive cells with labeling either with anti-neuron specific enolase or anti-parvalbumin (indicating GABAnergic interneurons) antibody spread in the deep cortical layers adjacent to the injury and co-localized with activated μ-calpain. Connexin (Cx)-43, glial fibrillary acidic protein (GFAP), activated μ-calpain and α-fodrin breakdown product (FBP) increased post-injury, peaking at 1 h, in the injury and adjacent areas. GFAP-Cx43-positive reactivated astrocytes exhibited similar distribution to the dead neurons. Cx43 and Cx36 primarily comprise HCs in the astrocyte and neuron, respectively. Ethidium bromide (EtBr) uptake was enhanced post-injury, and confirmed in the Cx43- and Cx36-positive cells. A Cx43-HC inhibitor Gap26 prevented the opening of the Cx43-HC and Cx36-HC, μ-calpain activation, α-fodrin proteolysis and death in the deep cortical neurons. Collectively, opening of the astrocytic Cx43-HC and neuronal Cx36-HC would induce the regional spread of cortical neuronal death through μ-calpain activation in the rat brain injury model.     

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.     

Functioning of cx43 hemichannels demonstrated by single channel properties

It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.     

Expression of gap junction protein connexin43 in the adult rat cochlea: comparison with connexin26.

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.