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.     

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.     

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

The effect of CO₂-induced acidification on transjunctional voltage (V_j) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO₂ application greatly decreased the V_j sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO₂ also affected the kinetics of V_j dependent inactivation of junctional current (I_j), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO₂ sensitivity of chemical gating such that even CO₂ concentrations as low as 2.5% significantly lowered junctional conductance (G_j). With Cx50 channels G_j dropped by 78% with a drop in intracellular pH (pH_i) to 6.83, whereas with Cx50-D3N channels G_j dropped by 95% with a drop in pH_i to just 7.19. We have previously hypothesized that the way in which V_j gating reacts to CO₂ might be related to connexin’s gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO₂ on V_j gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes     

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     

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.     

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.     

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

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

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.

     

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     

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.     

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.     

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.     

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.     

The First Extracellular Domain Plays an Important Role in Unitary Channel Conductance of Cx50 Gap Junction Channels

Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as V_j-gating) and display a wide range of unitary channel conductance (γ_j), yet the domains responsible for V_j-gating and γ_j are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in V_j-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct V_j-gating and γ_j from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γ_j, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in V_j-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in V_j-gating and γ_j. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.     

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.     

Calmodulin Colocalizes with Connexins and Plays a Direct Role in Gap Junction Channel Gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their V_j sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).     

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