Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Properties of gap junction channels formed by Cx46 alone and in combination with Cx50

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.     

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.     

Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V(j). Cx40/Cx45 heterotypic GJ channels preferentially exhibit V(j)-dependent gating transitions between open and residual states with a conductance of approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.     

Conductance and permeability of the residual state of connexin43 gap junction channels

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.     

Functional Characterization of Canine Connexin45

Three gap junctional proteins have been identified in canine ventricular myocytes: connexin 43 (Cx43), connexin 45 (Cx45), and connexin 40 (Cx40). We have characterized the functional properties of canine Cx45 and examined how Cx45 functionally interacts with Cx43 in Xenopus oocyte pairs. Homotypic pairs expressing Cx45 were well coupled. Heterotypic pairs composed of Cx45 paired with either Cx43 or Cx38 also developed high levels of conductance. Junctional currents in the heterotypic pairs displayed a highly asymmetrical voltage dependence. The kinetics and steady-state voltage dependence of the heterotypic channels more closely resembled those of the Cx45 channels when the Cx45 cRNA-injected cell was relatively negative suggesting that the Cx45 connexon closes for relative negativity at the cytoplasmic end of the channel. We also show that homotypic and heterotypic channels composed of Cx45 and Cx43 exhibit differences in pH _i sensitivity. Received: 18 August 1995/Revised: 21 November 1995     

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons

Recent evidence indicatingformation of functional homomeric/heterotypic gap junction channels byconnexin40 (Cx40) and connexin43 (Cx43) raises the question of whetherdata previously interpreted as support for heteromeric channelformation by these connexins might not instead reflect the activity ofhomomeric/heterotypic channels. To address this question and to furthercharacterize the behavior of these channels, we used dual whole cellvoltage-clamp techniques to examine the junctions formed between cellsthat express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired withcells that naturally express both connexins (A7r5 cells). Rin40/Rin43cell pairs formed functional gap junctions that displayed a stronglyasymmetric voltage-dependent gating response. Single-channel eventamplitudes ranged between 34 and 150 pS, with 90- to 130-pS eventspredominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairsdemonstrating strong asymmetry. These cell pairs exhibited a variety ofsingle-channel events that were not consistent with homomeric/homotypicCx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels.These data indicate that Cx40 and Cx43 form homomeric/heterotypic aswell as heteromeric/heterotypic channels that display unique gating andconductance properties.

     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.     

Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein.

We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.     

Cx40 and Cx43 expression ratio influences heteromeric/ heterotypic gap junction channel properties

Incells that coexpress connexin (Cx)40 and Cx43, the ratio of expressioncan vary depending on the cellular environment. We examined the effectof changing Cx40:Cx43 expression ratio on functional gap junctionproperties. Rin cells transfected with Cx40 or Cx43 (Rin40, Rin43) werecocultured with 6B5n, A7r5, A7r540C1, or A7r540C3 cells forelectrophysiological and dye coupling analysis. Cx40:Cx43 expressionratio in 6B5n, A7r5, A7r540C1, and A7r540C3 cells was ~1:1, 3:1, 5:1,and 10:1, respectively. When Rin43 cells were paired with coexpressingcells, there was an increasing asymmetry of voltage-dependent gatingand a shift toward smaller conductance events as Cx40:Cx43 ratioincreased in the coexpressing cell. These observations could not bepredicted by linear combinations of Cx40 and Cx43 properties inproportion to the expressed ratios of the two Cxs. When Rin40 cellswere paired with coexpressing cells, the net voltage gating andsingle-channel conductance behavior were similar to those ofRin40/Rin40 cell pairs. Dye permeability properties of cell monolayersdemonstrated that as Cx40:Cx43 expression ratio increased incoexpressing cells the charge and size selectivity of dye transferreflected that of Rin40 cells, as would be predicted. These dataindicate that the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents expressed in the cell; however, the dyepermeability of these same channels can be predicted by the relative Cx contributions.

     

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V _j-sensitive gating of I _j (V _j, gap junction voltage; I _j, gap junction current), (2) contribution and (3) kinetics of I _j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V _j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.     

Functional characteristics of heteromeric Cx40-Cx43 gap junction channel formation

Cx40:Cx43 expression ratio in A7r5 cells is augmented in growth stimulated vs. growth arrested conditions. To determine the impact of changing Cx40:Cx43 expression ratio on gap junction function, we have developed A7r5 cell lines that display Cx40:Cx43 ratios of 1:1 (66B5n) and 10:1 (A7r540C3). When Rin43 cells were paired with these coexpressing cells, there was an increasing asymmetry of voltage dependent gating as the Cx40:Cx43 ratio increased in the coexpressing cell. This asymmetry was opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In addition, when Rin43 cells were paired with coexpressing cells there was a shift toward smaller single channel event amplitudes with increasing Cx40:Cx43 ratio in the coexpressing cell. Again, this is opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In dye coupling experiments, 6B5N, A7r5, and A7r540C3 cells displayed charge and size selectivity that increased with increasing Cx40:Cx43 expression ratio. These data indicate that although the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents that comprise the channel, the dye permeability data fit what would be predicted by an increase in Cx40:Cx43 ratio.     

Functional Characteristics of Heteromeric Cx40-Cx43 Gap Junction Channel Formation

Cx40:Cx43 expression ratio in A7r5 cells is augmented in growth stimulated vs. growth arrested conditions. To determine the impact of changing Cx40:Cx43 expression ratio on gap junction function, we have developed A7r5 cell lines that display Cx40:Cx43 ratios of 1:1 (6B5n) and 10:1 (A7r540C3). When Rin43 cells were paired with these coexpressing cells, there was an increasing asymmetry of voltage dependent gating as the Cx40:Cx43 ratio increased in the coexpressing cell. This asymmetry was opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In addition, when Rin43 cells were paired with coexpressing cells there was a shift toward smaller single channel event amplitudes with increasing Cx40:Cx43 ratio in the coexpressing cell. Again, this is opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In dye coupling experiments, 6B5N, A7r5, and A7r540C3 cells displayed charge and size selectivity that increased with increasing Cx40:Cx43 expression ratio. These data indicate that although the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents that comprise the channel, the dye permeability data fit what would be predicted by an increase in Cx40:Cx43 ratio.     

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 ～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 ～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.     

Neonatal rat cardiomyocytes show characteristics of nonhomotypic gap junction channels

Neonatal rat cardiomyocytes mainly coexpress the connexins Cx40, Cx43, and to a small amount Cx45, leading to potential formation of mixed (heteromeric/heterotypic) gap junction channels. Using the dual-voltage clamp technique with switching clamp circuits, the authors investigated voltage sensitivity of gap junction channels between cell pairs of Cx40, Cx43, and Cx45 stably transfected HeLa cells and compared those data to data obtained from cell pairs of cultured neonatal rat cardiomyocytes. In accordance to previously published data, the relationship between normalized conductance and transjunctional voltage (g/V(j)) was quasisymmetrical for the transfected HeLa cells, indicating homotypic gap junction channels. Boltzmann curves fitted to data obtained from neonatal rat cardiomyocyte pairs expressing both Cx40 and Cx43 showed an asymmetrical inactivation pattern, which cannot be explained by the presence of pure populations of homotypic gap junction channels of either isoform. In conclusion the authors assume the additional presence of heterotypic and possibly even heteromeric gap junction channels in neonatal rat cardiomyocytes.     

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.     

Molecular dissection of transjunctional voltage dependence in the connexin-32 and connexin-43 junctions.

Most gap junction channels are sensitive to the voltage difference between the two cellular interiors, termed the transjunctional voltage (V(j)). In several junctions, the conductance transitions induced by V(j) show more than one kinetic component. To elucidate the structural basis of the fast and slow components that characterize the V(j )dependence of connexin-32 (Cx32) and connexin-43 (Cx43) junctions, we created deletions of both connexins, where most of the carboxy-terminal (CT) domain was removed. The wild-type and "tailless" mutants were expressed in paired Xenopus oocytes, and the macroscopic gating properties were analyzed using the dual voltage clamp technique. Truncation of the CT domain of Cx32 and Cx43 abolished the fast mechanism of conductance transitions and induced novel gating properties largely attributable to the slow mechanism of gating. The formation of hybrid junctions comprising wild-type and truncated hemichannels allowed us to infer that the fast and slow components of gating reside in each hemichannel and that both gates close at a negative V(j) on the cytoplasmic side. Thus we conclude that the two kinetic components of V(j)-sensitive conductance are a result of the action of two different gating mechanisms. They constitute separate structures in the Cx32 and Cx43 molecules, the CT domain being an integral part of fast V(j) gating.