Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels

Connexinchannels are gated by transjunctional voltage(V_j)or CO₂ via distinct mechanisms.The cytoplasmic loop (CL) and arginines of a COOH-terminal domain(CT₁) of connexin32 (Cx32) wereshown to determine CO₂sensitivity, and a gating mechanism involvingCL-CT₁ association-dissociationwas proposed. This study reports that Cx32 mutants, tandem, 5R/E, and5R/N, designed to weaken CL-CT₁interactions, display atypicalV_jand CO₂ sensitivities when testedheterotypically with Cx32 wild-type channels inXenopus oocytes. In tandems, two Cx32monomers are linked NH₂-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replaceCT₁ arginines. On the basis of theintriguing sensitivity of the mutant-32 channel toV_jpolarity, the existence of a "slow gate" distinct from theconventionalV_jgate is proposed. To a lesser extent the slow gatemanifests itself also in homotypic Cx32 channels. Mutant-32 channelsare more CO₂ sensitive than homotypic Cx32 channels, andCO₂-induced chemical gating isreversed with relative depolarization of the mutant oocyte, suggesting V_jsensitivity of chemical gating. A hypothetical pore-plugging modelinvolving an acidic cytosolic protein (possibly calmodulin) is discussed.

     

The Voltage Gates of Connexin Channels are Sensitive to CO2

Cx45 channel sensitivity to CO₂, transjunctional voltage (V_j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage-clamp. Cx45 channels are very sensitive to V_jand close preferentially by the slow gate, likely the same as the chemical gate. With CO₂-induced drop in junctional conductance (G_j), the speed of V_j-dependent inactivation of junctional current (I_j) and V_jsensitivity increased. With 40 mV V_j, the τ of single exponential I_jdecay reversibly decreased by ～40% with CO₂, and G_{j steady state}/G_{j peak}decreased multiphasically, indicating that kinetics and V_jsensitivity of chemical/slow-V_jgating are altered by changes in [H⁺]_iand/or [Ca²⁺]_i. With 15 min exposure to CO₂, G_jdropped to 0% in controls and by ～17% following CaM expression inhibition; similarly, V_jsensitivity decreased significantly. This indicates that the speed and sensitivity of V_j-dependent inactivation of Cx45 channels are increased by CO₂, and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G_{j steady state}/G_{j peak}and τ with CO₂matched more closely that of G_{j peak}. In contrast, sensitivity and speed of V_jgating of Cx40 and Cx26 channels decreased, rather than increased, with CO₂application.     

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.     

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.     

CO<Subscript>2</Subscript> Sensitivity of Voltage Gating and Gating Polarity of GapJunction Channels—Connexin40 and its COOH-Terminus-Truncated Mutant

The CO₂ sensitivity of transjunctional voltage (V _j) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V _j sensitivity, determined by a standard V _j protocol (20 mV V _j steps, 120 mV maximal), decreased significantly with exposure to 30% CO₂. The Boltzmann values of control versus CO₂-treated oocytes were: V ₀ = 36.3 and 48.7 mV, n = 5.4 and 3.7, and G _{j min} = 0.21 and 0.31, respectively. CO₂ also affected the kinetics of V _j-dependent inactivation of junctional current (I _j); the time constants of two-term exponential I _j decay, measured at V _j = 60 mV, increased significantly with CO₂ application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO₂ was also unaffected by CT truncation. There is evidence that CO₂ decreases the V _j sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side of V _j, whereas Cx45 and Cx32 gate at negative V _j, it is likely that V _j behavior with respect to CO₂-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V _j sensor (NH₂-terminus).This revised version was published online in June 2005 with a corrected cover date.     

Is the Voltage Gate of Connexins CO<Subscript>2</Subscript>-sensitive? Cx45 Channels and Inhibition of Calmodulin Expression

The sensitivity of Cx45 channels to CO₂, transjunctional voltage (V _j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage clamp. Cx45 channels are very sensitive to V _j and close with V _j preferentially by the slow gate, likely to be the same as the chemical gate. With a CO₂-induced drop in junctional conductance (G _j), both the speed of V _j-dependent inactivation of junctional current (I _j) and V _j sensitivity increased. With 40-mV V _j-pulses, the of single exponential I _j decay reversibly decreased by 40% during CO₂ application, and G_{j steady state}/G_{j peak} decreased multiphasically, indicating that both kinetics and V _j sensitivity of chemical/slow V _j gating are altered by changes in [H⁺]_i and/or [Ca²⁺]_i. CaM expression was inhibited with oligonucleotides antisense to CaM mRNA. With 15 min CO₂, relative junctional conductance (G _jt/G _jt0) dropped to 0% in controls, but only by 17% in CaM-antisense oocytes. Similarly, V _j sensitivity was significantly lessened in CaM-antisense oocytes. The data indicate that both the speed and sensitivity of V _j-dependent inactivation of the junctional current of Cx45 channels are affected by CO₂ application, and that CaM plays a key role in channel gating.     

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     

Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity

Connexin32(Cx32) mutants were studied by double voltage clamp inXenopus oocytes to determine the roleof basic COOH-terminal residues in gap junction channel gating byCO₂ and transjunctional voltage.Replacement of five arginines with N (5R/N) or T residues in theinitial COOH-terminal domain(CT₁) of Cx32 enhancedCO₂ sensitivity. The positivecharge, rather than the R residue per se, is responsible for theinhibitory role of CT₁, becausemutants replacing the five R residues with K (5R/K) or H (5R/H)displayed CO₂ sensitivitycomparable to that of wild-type Cx32. Mutants replacing R with Nresidues four at a time (4R/N) showed that CO₂ sensitivity is stronglyinhibited by R215 and mildly by R219, whereas R220, R223, and R224 mayslightly increase sensitivity. Neither the 5R/N nor the 4R/N mutantsdiffered in voltage sensitivity from wild-type Cx32. The possibilitythat inhibition of gating sensitivity results from electrostaticinteractions between CT₁ and thecytoplasmic loop is discussed as part of a model that envisions thecytoplasmic loop of Cx32 as a key element of chemical gating.

     

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 ± 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage (G_j-V_j) relationship was symmetrical and well described by a two-state Boltzmann relation (G_min 0.33, V₀ = 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was 120 pS for the fully open channel and 26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing V_j, accounting for the V_j-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified. bladder function; intercellular communication; smooth muscle     

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.     

Unusual Slow Gating of Gap Junction Channels in Oocytes Expressing Connexin32 or Its COOH-Terminus Truncated Mutant

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V _j)-sensitive gates: fast and slow. Slow V _j gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V _j and is mostly inactive without uncouplers or connexin (Cx) mutations. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of −100 mV V _j pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G _j), measured at each pulse, decreased exponentially by 50−60% (tau = ∼1.2 min). G _jPK dropped more dramatically, such that G _jSS/G _jPK increased from 0.4 to 0.6, indicating a drop in V _j sensitivity. Less striking effects were obtained with –60 mV pulses. During recovery, G _j, measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = ∼7 min). With reversal of V _j polarity, G _jPK briefly increased and G _jSS/G _jPK decreased, suggesting that V _j-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V _j gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V _j behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.     

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     

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.     

Biophysical characterization of zebrafish connexin35 hemichannels

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (I_hc) that were activated and increased by stepwise depolarization of membrane potential (V_m) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (_hc: 250–320 pS). The _hc of Cx35 hemichannels exhibited a pronounced V_m dependence; i.e., _hc increased/decreased with relative hyperpolarization/depolarization (_hc was 72 pS at V_m = –100 mV and 35 pS at V_m = 100 mV). Extrapolation to V_m = 0 mV predicted a _hc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of 24 pS. Channel gating was also V_m dependent: open time declined with negative V_m and increased with positive V_m. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo. connexin; gating; retina     

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.     

The Carboxyl Terminal Residues 220–283 Are Not Required for Voltage Gating of a Chimeric Connexin32 Hemichannel

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of V_j- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the V_j (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and V_j gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32^∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both V_j- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR²¹⁹) of the Cx32^∗43E1 hemichannel. We conclude that an operational Cx32 V_j (fast) gate does not require CT residues 220–283, as reported previously by others.     

The Carboxyl Terminal Residues 220–283 Are Not Required for Voltage Gating of a Chimeric Connexin32 Hemichannel

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of V_j- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the V_j (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and V_j gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32^∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both V_j- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR²¹⁹) of the Cx32^∗43E1 hemichannel. We conclude that an operational Cx32 V_j (fast) gate does not require CT residues 220–283, as reported previously by others.     

Exchange of gating properties between rat cx46 and chicken cx45.6

Cx46 and Cx50 are coexpressed in lens fiber cells where they form fiber-fiber gap junctions. Recent studies have shown that both proteins play a critical role in maintaining lens transparency. Although both Cx46 and Cx50 (or its chicken ortholog, Cx45.6) show a high degree of sequence homology, they exhibit marked differences in gap junctional channel gating, unitary gap junctional channel conductance, and hemichannel gating. To better understand which regions of the protein are responsible for these functional differences, we have constructed a series of chimeric Cx46-Cx45.6 gap junctional proteins in which a single transmembrane or intracellular domain of Cx45.6 was replaced with the corresponding domain of Cx46, expressed them in Xenopus oocyte pairs or N2A cells, and examined the resulting gap junctional conductances. Our results showed that four out of six of the chimeras induced high levels of gap junctional coupling. Of these chimeras, only Cx45.6-46NT showed significant changes in voltage-dependent gating properties. Exchanging the N-terminus had multiple effects. It slowed the inactivation kinetics of the macroscopic junctional currents so that they resembled those of Cx46, reduced the voltage sensitivity of the steady-state junctional conductance, and decreased the conductance of single gap junctional channels. Additional point mutations identified a uniquely occurring arginine in the N-terminus of Cx46 as the main determinant for the change in voltage-dependent gating.     

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