Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels.

The gap-junctional protein rat connexin46 (Cx46) has the unusual ability to form voltage-gated channels in the nonjunctional plasma membrane of Xenopus oocytes (Paul et al., 1991; Ebihara and Steiner, 1993). These have been suggested to be gap-junctional hemichannels or connexons. The Xenopus oocyte system was used to characterize the functional properties of a closely related lens gap-junctional protein, chicken connexin56 (Cx56) (Rup et al., 1993) and to contrast them to those of rat Cx46. Single oocytes injected with either Cx56 or Cx46 cRNA developed time-dependent, outward currents that activated on depolarization. The currents induced by Cx56 and Cx46 showed differences in steady-state voltage dependence and in their degree of rectification. Furthermore, the voltage-dependent properties of the nonjunctional channels induced by the connexin cRNAs in external solutions containing low concentrations of calcium ions could account remarkably well for the behavior of the intercellular channels formed by Cx56 and Cx46 in paired oocytes. These results suggest that many of the voltage-dependent properties of the hemi-gap-junctional channels are retained by the intercellular channels.     

Inhibition of Gap Junction Hemichannels by Chloride Channel Blockers

Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.     

Functional expression in Xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43

Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.     

Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor

Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.     

Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera

Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95-117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733-779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Regulation of connexons composed of human connexin26 (hCx26) by temperature

This report shows that temperature is a latent regulator of the voltage-dependent conductance of hemichannels composed of hCx26. The latter were expressed in Xenopus oocytes by injection of a mixture of hCx26 cRNA and antisense of endogenous Cx38 (anti-Cx38). At 24-25 degrees C, voltage clamp of oocytes at potentials above -40 mV evoked outward currents which were not observed in control oocytes. These currents were reversibly affected by change in temperature. Increasing temperature of the bath solution amplified gradually, whereas decreasing bath temperatures below 20 degrees C reduced the current. Furthermore analysis revealed that temperature-dependent increase of the conductance of the hemichannels did not correlate with a change of the apparent gating charge, whereas the half-activation voltage V(1/2) of the hemichannel was affected by a temperature change. It is proposed that this finding correlates with a temperature-dependent transition into an open state above 20 degrees C. In addition, a temperature-dependent release of Lucifer Yellow from loaded liposomes containing reconstituted purified and hCx26 hemichannels was observed, which indicate that a temperature-dependent regulation of the permeability of hCx26 hemichannels is not related to intracellular mediators. The involvement of temperature to modulate hemichannels as well as of the corresponding gap junction channel composed of hCx26 at physiological condition is discussed.     

Distinct permeation profiles of the connexin 30 and 43 hemichannels

Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate, lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes. C-terminally truncated Cx43 (M257) displayed increased dye uptake and, unlike wild type Cx43 channels, conducted current. Neither Cx30 nor Cx43 acted as water channels in their hemichannel configuration. Our results demonstrate that connexin hemichannels have isoform-specific permeability profiles and that dye uptake cannot be equaled to permeability of smaller physiologically relevant molecules in given settings.     

Virtual cloning,functional expression,and gating analysis of human connexin31.9

We have identified a novel gap junctiongene by searching the human genome sequence database that encodes aprotein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologousto human Cx32.4 and did not cluster with either the purported - or-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCRin human mRNA from several tissues including cerebral cortex, heart,liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by anapparent low unitary conductance (12-15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channelswere gated by cytoplasmic acidification or exposure to halothane likeother connexins. Cx31.9 was able to form heterotypic channels with thehighly voltage-sensitive Xenopus Cx38 (XenCx38), whichprovides an opportunity to study gating in heterotypic channels formedby hemichannels (connexons) composed of connexins with widely divergentproperties. Thus Cx31.9 is a novel human connexin that forms channelswith unique functional properties.

     

Implication of gap junction coupling in amphibian vitellogenin uptake

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca²⁺ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.     

Connexin 48.5 is required for normal cardiovascular function and lens development in zebrafish embryos

Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric mixing of connexins: compatibility of partners and functional consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.     

Xenopus connexin38 forms hemi-gap-junctional channels in the nonjunctional plasma membrane of Xenopus oocytes.

A nonselective cation current activated by depolarization (Ic) is present in the nonjunctional membrane of Xenopus oocytes. This current shares a number of properties with hemi-gap-junctional currents induced by exogenous gap-junctional proteins in oocytes and with a nonjunctional current seen in teleost retinal horizontal cells including nonselective permeability to small cations, block by external divalent cations, and slow activation kinetics. Here we study the effects of depleting or overexpressing Cx38 on Ic. Antisense depletion of Cx38 caused a marked reduction in Ic and blocked endogenous gap-junctional coupling in oocyte pairs. Conversely, expression of cloned Cx38 in oocytes increased the amplitude of Ic and enhanced gap-junctional coupling. Furthermore, there appeared to be a close correlation between the temperature sensitivity of Ic and the temperature sensitivity of assembly of endogenous gap-junctional channels in oocyte pairs. These results suggest that Xenopus connexin38 is involved in the generation of Ic.