Pore-lining residues identified by single channel SCAM studies in Cx46 hemichannels.

The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify residues that participate in lining the aqueous pore of channels formed of connexins. Criteria for assignment to the pore included reactivity to sulfydryl-specific methanethiosulfonate (MTS) reagents from both sides of an open hemichannel and observable effects on open channel properties. We demonstrate reactivity to MTS reagents over a stretch of seventeen amino acids, D51 through L35, that constitute segments of E1 and TM1. Qualitatively, the nature of the effects caused by the Cys substitutions alone and their modification with MTS reagents of either charge indicate side chain valence is most influential in determining single channel properties with D51 and L35 defining the extracellular and intracellular limits, respectively, of the identified pore-lining region. A number of Cys substitutions beyond L35 in TM1 caused severe alterations in hemichannel function and precluded assignment to the pore. Although all six subunits can be modified by smaller MTS reagents, modifications appear limited to fewer subunits with larger reagents.

Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.

Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

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

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

Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease.

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons.

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels.

Intercellular channels formed of members of the gene family of connexins (Cxs) vary from being substantially cation selective to being anion selective. We took advantage of the ability of Cx46 to function as an unopposed hemichannel to examine the basis of Cx charge selectivity. Previously we showed Cx46 hemichannels to be large pores that predominantly conduct cations and inwardly rectify in symmetric salts, properties suggesting selectivity is influenced by fixed negative charges located toward the extracellular end of the pore. Here we demonstrate that high ionic strength solutions applied to the extracellular, but not the intracellular, side of Cx46 hemichannels substantially reduce the ratio of cation to anion permeability. Substitution of the first extracellular loop (E1) domain of Cx32, an anion-preferring Cx, reduces conductance, converts Cx46 from cation to anion preferring, and changes the I-V relation form inwardly to outwardly rectifying. These data suggest that fixed negative charges influencing selectivity in Cx46 are located in E1 and are substantially reduced and/or are replaced with positive charges from the Cx32 E1 sequence. Extending studies to Cx46 cell-cell channels, we show that they maintain a strong preference for cations, have a conductance nearly that expected by the series addition of hemichannels, but lack rectification in symmetric salts. These properties are consistent with preservation of the fixed charge region in E1 of hemichannels, which upon docking, become symmetrically placed near the center of the cell-cell channel pore. Furthermore, heterotypic cell-cell channels formed by pairing Cx46 with Cx32 or Cx43 rectify in symmetric salts in accordance with the differences in the charges we ascribed to E1. These data are consistent with charged residues in E1 facing the channel lumen and playing an important role in determining Cx channel conductance and selectivity.

Structure of the amino terminus of a gap junction protein.

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.

Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32.

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed "V(j)-gating" and "loop gating" (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351;. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.

Functional expression of the murine connexin 36 gene coding for a neuron-specific gap junctional protein.

The mouse connexin 36 (Cx36) gene was mapped on chromosome 2 and an identical transcriptional start site was determined in brain and retina on exon I. Rabbit polyclonal antibodies to the presumptive cytoplasmic loop of the Cx36 protein recognized in immunohistochemical analyses Cx36 expression in the retina, olfactory bulb, hippocampus, inferior olive and cerebellum. In olivary neurons strong punctate labeling at dendritic cell contacts and weaker labeling in the cytoplasm of dendrites were shown by immuno electron microscopy. After expression of mouse Cx36 cDNA in human HeLa cells, neurobiotin transfer was increased 1.8-fold and electrical conductance at least 15-fold compared to untransfected HeLa cells. No Lucifer Yellow transfer was detected in either untransfected or Cx36 transfected HeLa cells. Single Cx36 channels in transfected HeLa cells showed a unitary conductance of 14.3 + or - 0. 8 pS. The sensitivity of Cx36 channels to transjunctional voltage was low in both HeLa-Cx36 cells and Xenopus oocytes expressing mouse Cx36. No increased transfer of neurobiotin was detected in heterotypic gap junctions formed by Cx36 and 9 other connexins expressed in HeLa cells. Our results suggest that Cx36 channels function as electrical synapses for transmission of electrical and metabolic signals between neurons in the central nervous system.

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.

Connexin hemichannels and cell-cell channels: comparison of properties.

Connexin46 (Cx46) forms functional hemichannels in the absence of contact by an apposed hemichannel and we have used these hemichannels to study gating and permeation at the single channel level with high time resolution. Using both cell-attached and -excised patch configurations, we find that single Cx46 hemichannels exhibit some properties expected of half of a gap junction channel, as well as novel properties. Cx46 hemichannels have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to TEA. Both monovalent cations and anions can permeate, but cations are substantially more permeable. The open channel conductance shows marked inward rectification in symmetric salts. We find that the conductance and permeability properties of Cx46 cell-cell channels can be explained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are conserved. Also like cell-cell channels, unapposed Cx46 hemichannels are closed by elevated levels of H+ or Ca2+ ions on the cytoplasmic face. Closure occurs in excised patches indicating that the actions of these agents do not require a soluble cytoplasmic factor. Fast (<0.5 ms) application of H+ to either side of the open hemichannel causes an immediate small reduction in unitary conductance followed by complete closure with latencies that are dependent on H+ concentration and side of application; sensitivity is much greater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not require that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hemichannels, but at high enough concentration close them by acting at a cytoplasmic site(s) that causes a conformational change resulting in complete closure. Extracellular H+ may permeate to act on the cytoplasmic site or act on a lower affinity extracellular site. Thus, the unapposed hemichannel is a valuable tool in addressing fundamental questions concerning the operation of gap junction channels that are difficult to answer by existing methods. The ability of Cx46, and perhaps other connexins, to form functional unapposed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasma membrane.

Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells.

Communication-incompetent cell lines were transfected with connexin (Cx) 43 fused with enhanced green fluorescent protein (EGFP) to examine the relation between Cx distribution determined by fluorescence microscopy and electrical coupling measured at single-channel resolution in living cell pairs. Cx43-EGFP channel properties were like those of wild-type Cx43 except for reduced sensitivity to transjunctional voltage. Cx43-EGFP clustered into plaques at locations of cell-cell contact. Coupling was always absent in the absence of plaques and even in the presence of small plaques. Plaques exceeding several hundred channels always conferred coupling, but only a small fraction of channels were functional. These data indicate that clustering may be a requirement for opening of gap junction channels.

Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32.

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current-voltage (I-V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current-voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I-V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405-1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I-V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I-V relations that closely resembled the observed I-V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (V(j))-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.

Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.

pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.

Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease.

The relationship between the loss of connexin 32 function and clinical manifestations of X-linked Charcot-Marie-Tooth (CMTX) disease is unknown. Here, we report that eight of nine CMTX mutations investigated form channels with measurable electrical conductance. Single-channel studies of two mutations demonstrate reduced junctional permeability caused by a decrease in either pore size (S26L) or open channel probability (M34T) that favors residency in a low-conductance substate. Permeation of second messengers such as cAMP through reflexive gap junctions between adjacent cytoplasmic loops of myelinating Schwann cells is likely to be reduced or absent in these channels. We propose that CMTX mutations impair the transduction of signals arising from normal glial-neuronal interactions and thereby cause demyelination and axonal degeneration.

Voltage gating and permeation in a gap junction hemichannel.

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.

Opposite voltage gating polarities of two closely related connexins.

The molecular mechanisms underlying the voltage dependence of intercellular channels formed by the family of vertebrate gap junction proteins (connexins) are unknown. All vertebrate gap junctions are sensitive to the voltage difference between the cells, defined as the transjunctional voltage, Vj (refs 1, 2), and most appear to gate by the separate actions of their component hemichannels. The heterotypic Cx32/Cx26 junction displays an unpredicted rectification that was reported to represent a novel Vj dependence created by hemichannel interactions, mediated in part by the first extracellular loop E1 (ref. 9). Here we show that aspects of the rectification of Cx32/Cx26 junctions are explained by opposite gating polarities of the component hemichannels, and that the opposite gating polarity of Cx32 and Cx26 results from a charge difference in a single amino-acid residue located at the second position in the N terminus. We also show that charge substitutions at the border of the first transmembrane (M1) and E1 domains can reverse gating polarity and suppress the effects of a charge substitution at the N terminus. We conclude that the combined actions of residues at the N terminus and M1/E1 border form a charge complex that is probably an integral part of the connexin voltage sensor. A consistent correlation between charge substitution and gating polarity indicates that Cx26 and Cx32 voltage sensors are oppositely charged and that both move towards the cytoplasm upon hemichannel closure.