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.     

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.     

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.     

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.

     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻ channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G _j) and its sensitivity to transjunctional voltage (V _j) by the dual voltage-clamp method. Application of forskolin induced a Cl⁻ current; increased G _j approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V _j gating, monitored by a change in the ratio between G _j steady state and G _j peak (G _jSS/G _jPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G _j and decreased the sensitivity to V _j gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G _j and V _j changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_jand γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_jin Cx40/Cx40 pairs, but decreased g_jin the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

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.     

The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V_j-gating) and the unitary gap junction channel conductance (γ_j). To better understand the molecular structure(s) determining the V_j-gating properties and the γ_j of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to V_j (G_j/V_j relationship), slowed V_j-gating kinetics, and reduced γ_j as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V_j-gating properties and γ_j to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The G_j/V_j relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (G_min) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (γ_j = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V_j-gating and the γ_j, and the introduction of a positively charged Arg at the ninth position reduced the G_min with a correlated disappearance of the substate at the single channel level.     

A Stochastic Four-State Model of Contingent Gating of Gap Junction Channels Containing Two “Fast” Gates Sensitive to Transjunctional Voltage

Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two “fast” gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (V_j). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance γ_h,o and in the residual state with conductance γ_h,res, and that both γ_h,o and γ_h,res rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of V_j that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by γ_h,o and γ_h,res rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and V_j-dependent gating of GJs formed from different connexin isoforms. V_j-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive V_j steps rising in amplitude, 2), slowly rising V_j ramps, and 3), series of V_j steps of high frequency. The model was used to predict V_j-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of V_j-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels.     

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     

Chemical Gating of Heteromeric and Heterotypic Gap Junction Channels

Gap junction channels contain two hemichannels (connexons), each being a connexin (Cx) hexamer. In cells expressing multiple connexins, heteromeric connexons are believed to form, whereas cell pairs expressing different connexins generate heterotypic channels. To define gating behavior of heteromeric and heterotypic channels, CO₂-induced gating was tested in Xenopus oocyte pairs expressing Cx32, or 5R/N (Cx32 mutant), as well as in pairs in which one oocyte (mx) expressed a 50/50 mixture of Cx32 and 5R/N and the other either the mixture (mx), Cx32 (32) or 5R/N (R/N). In 5R/N, replacement of 5 C-terminus arginines with asparagines greatly increased CO₂ sensitivity. In response to 3 and 15 min CO₂ exposures, junctional conductance (G _j ) decreased to 85% and 47%, in 32–32 pairs, and to 7% and 0.9%, in R/N-R/N pairs, respectively. In mx-mx and mix-32 pairs, G _j decreased to similar values (33% and 35%, respectively) with 15 min CO₂. The sensitivity of mx-R/N pairs was similar to that of heterotypic 32-R/N pairs, as G _j dropped to 36% and 38%, respectively, with 3 min CO₂. Monoheteromeric (mx-32 and mx-R/N) and biheteromeric (mx-mx) channels behaved as if Cx32 were dominant, suggesting that hemichannel sensitivity is not an average of the sensitivities of its connexin monomers. In contrast, heterotypic channels behaved as if the two hemichannels of a cell-cell channel had no influence on each other. Received: 15 May 1997/Revised: 8 December 1997     

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

Homomeric gap junction channels are composed solely of oneconnexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different fromeach other. A heteromeric gap junction channel is one that containsdifferent connexins within either or both hemichannels. The existenceof heteromeric forms has been suggested, and many cell types are knownto coexpress connexins. To determine if coexpressed connexins wouldform heteromers, we cotransfected rat connexin43 (rCx43) and humanconnexin37 (hCx37) into a cell line normally devoid of any connexinexpression and used dual whole cell patch clamp to compare the observedgap junction channel activity with that seen in cells transfected onlywith rCx43 or hCx37. We also cocultured cells transfected with hCx37 orrCx43, in which one population was tagged with a fluorescent marker tomonitor heterotypic channel activity. The cotransfected cells possessedchannel types unlike the homotypic forms of rCx43 or hCx37 or theheterotypic forms. In addition, the noninstantaneous transjunctionalconductance-transjunctional voltage(G_j/V_j)relationship for cotransfected cell pairs showed a large range ofvariability that was unlike that of the homotypic or heterotypic form.The heterotypic cell pairs displayed asymmetric voltage dependence. Theresults from the heteromeric cell pairs are inconsistent with summedbehavior of two independent homotypic populations or mixed populationsof homotypic and heterotypic channels types. TheG_j/V_jdata imply that the connexin-to-connexin interactions are significantlyaltered in cotransfected cell pairs relative to the homotypic andheterotypic forms. Heteromeric channels are a population of channelswhose characteristics could well impact differently from theirhomotypic counterparts with regard to multicellular coordinatedresponses.

     

Opposite Cx32 and Cx26 Voltage-Gating Response to CO2 Reflects Opposite Voltage-Gating Polarity

Previous studies have shown that the V_j-dependent gating behavior of gap junction channels is altered by CO₂ exposure. V_j-dependent channel closure is increased by CO₂ in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V_j (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO₂ affects V_j closure. To test this hypothesis, we have studied the CO₂-mediated changes in V_j gating in channels made of Cx32, Cx26, or a Cx32 mutant (Cx32-N2D) in which asparagine (N) at position 2 was replaced with aspartate (D). With exposure to CO₂, Cx32 channels (negative gaters) show increased V_j-dependent closure, whereas Cx26 channels (positive gaters) respond in the opposite way to V_j. Additionally, Cx32-N2D channels (positive gaters) show decreased V_j closure with exposure to CO₂. The reciprocal Cx26 mutant, Cx26-D2N (negative gater), could not be tested because it did not express functional homotypic channels. The data support the hypothesis that polarity of fast V_j gating determines whether CO₂ increases or decreases the V_j dependent closure of gap junction channels.     

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts. gap junctions; hearing; fluorescence resonance energy transfer; fluorescence recovery after photobleaching; immunoprecipitation; dye transfer; electrophysiology     

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.     

Voltage Gating of Gap Junctions in Cochlear Supporting Cells: Evidence for Nonhomotypic Channels

The organ of Corti has been found to have multiple gap junction subunits, connexins, which are localized solely in nonsensory supporting cells. Connexin mutations can induce sensorineural deafness. However, the characteristics and functions of inner ear gap junctions are not well known. In the present study, the voltage-dependence of gap junctional conductance (G _j ) in cochlear supporting cells was examined by the double voltage clamp technique. Multiple types of asymmetric voltage dependencies were found for both nonjunctional membrane voltage (V _m ) and transjunctional (V _j ) voltage. Responses for each type of voltage dependence were categorized into four groups. The first two groups showed rectification that was polarity dependent. The third group exhibited rectification with either voltage polarity, i.e., these cells possessed a bell-shaped G _j -V _j or G _j -V _m function. The rectification due to V _j had fast and slow components. On the other hand, V _m -dependent gating was fast (<5 msec), but stable. Finally, a group was found that evidenced no voltage dependence, although the absence of V _j dependence did not preclude V _m dependence and vice versa. In fact, for all groups V _j sensitivity could be independent of V _m sensitivity. The data show that most gap junctional channels in the inner ear have asymmetric voltage gating, which is indicative of heterogeneous coupling and may result from heterotypic channels or possibly heteromeric configurations. This heterogeneous coupling implies that single connexin gene mutations may affect the normal physiological function of gap junctions that are not limited to homotypic configurations. Received: 17 September 1999/Revised: 12 January 2000     

Functional analysis of selective interactions among rodent connexins.

One consequence of the diversity in gap junction structural proteins is that cells expressing different connexins may come into contact and form intercellular channels that are mixed in connexin content. We have systematically examined the ability of adjacent cells expressing different connexins to communicate, and found that all connexins exhibit specificity in their interactions. Two extreme examples of selectivity were observed. Connexin40 (Cx40) was highly restricted in its ability to make heterotypic channels, functionally interacting with Cx37, but failing to do so when paired with Cx26, Cx32, Cx43, Cx46, and Cx50. In contrast, Cx46 interacted well with all connexins tested except Cx40. To explore the molecular basis of connexin compatibility and voltage gating, we utilized a chimera consisting of Cx32 from the N-terminus to the second transmembrane domain, fused to Cx43 from the middle cytoplasmic loop to the C-terminus. The chimeric connexin behaved like Cx43 with regard to selectivity and like Cx32 with regard to voltage dependence. Taken together, these results demonstrate that the second but not the first extracellular domain affects compatibility, whereas voltage gating is strongly influenced by sequences between the N-terminus and the second transmembrane domain.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.