RESUMO
The permeability of gap junctions to tetraethylammonium ions was measured in isolated pairs of blastomeres from Rana pipiens L. and compared to the junctional conductance. In this system, the junctional conductance is voltage-dependent and decreases with moderate transjunctional voltage of either sign. The permeability to tetraethylammonium ions was determined by injecting one cell of a pair with tetraethylammonium and monitoring its changing concentration in the prejunctional and postjunctional cells with ion-selective electrodes. Junctional conductance was determined by current-clamp and voltage-clamp techniques. For different cell pairs in which the transjunctional voltage was small and the junctional conductance at its maximum value, the permeability to tetraethylammonium ions was proportional to the junctional conductance. In individual cell pairs, a reduction in the junctional conductance induced by voltage was accompanied by a proportional reduction in the permeability of the gap junction over a wide range. The diameter of the tetraethylammonium ion (8.0 to 8.5 A, unhydrated) is larger than that of the potassium ion (4.6 A, hydrated), the predominant current-carrying species. The proportionality between the permeability to tetraethylammonium ions and the junctional conductance, measured here with exceptionally fine time resolution, indicates that a common gap junctional pathway mediates both electrical and chemical fluxes between cells, and that closure of single gap junction channels by voltage is all or none.
Assuntos
Blastômeros/fisiologia , Junções Intercelulares/fisiologia , Animais , Comunicação Celular , Permeabilidade da Membrana Celular , Potenciais da Membrana , Rana pipiens , Tetraetilamônio , Compostos de Tetraetilamônio/fisiologiaRESUMO
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.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Mutação Puntual , Cromossomo X , Animais , Linhagem Celular , Conexinas/química , Condutividade Elétrica , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Oócitos/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetrodotoxina/farmacologia , Transfecção , Xenopus laevis , Proteína beta-1 de Junções ComunicantesRESUMO
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.
Assuntos
Encéfalo/metabolismo , Conexinas/biossíntese , Junções Comunicantes/metabolismo , Neurônios/metabolismo , Medula Espinal/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , Conexinas/genética , Corantes Fluorescentes , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Oócitos/citologia , Oócitos/metabolismo , Especificidade de Órgãos , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Medula Espinal/citologia , Transfecção , XenopusRESUMO
Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Cálcio/fisiologia , Coração/fisiologia , Junções Intercelulares/fisiologia , Animais , Cálcio/metabolismo , Dióxido de Carbono/farmacologia , Técnicas In Vitro , Masculino , Miocárdio/metabolismo , RatosRESUMO
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.
Assuntos
Conexinas/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Animais , Conexinas/química , Canais Iônicos/química , Potenciais da Membrana/fisiologia , Proteínas Recombinantes de Fusão/química , Xenopus , Proteína beta-1 de Junções ComunicantesRESUMO
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.
Assuntos
Conexinas/metabolismo , Cisteína/química , Espaço Extracelular/química , Animais , Cisteína/metabolismo , Eletrofisiologia , Espaço Extracelular/metabolismo , Indicadores e Reagentes , Canais Iônicos/metabolismo , Cinética , Potenciais da Membrana/fisiologia , Mesilatos , Oócitos/metabolismo , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/química , XenopusRESUMO
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.
Assuntos
Conexinas/fisiologia , Junções Comunicantes/fisiologia , Animais , Células Cultivadas , Conexina 26 , Conexinas/genética , Eletrofisiologia , Espaço Extracelular/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Camundongos , Modelos Neurológicos , Oócitos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/fisiologia , Sinapses/fisiologia , Transfecção , Proteína beta-1 de Junções ComunicantesRESUMO
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.
Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Algoritmos , Animais , Cálcio/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular , Citoplasma/metabolismo , Eletrofisiologia , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Xenopus laevisRESUMO
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.
Assuntos
Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Oócitos/metabolismo , Xenopus laevis/metabolismo , Substituição de Aminoácidos , Animais , Conexinas/metabolismo , Mesilatos/química , Modelos Moleculares , Técnicas de Patch-Clamp , Conformação ProteicaRESUMO
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.
Assuntos
Substituição de Aminoácidos , Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Junções Comunicantes/fisiologia , Mutação de Sentido Incorreto , Cromossomo X/genética , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Conexinas/química , Conexinas/fisiologia , Feminino , Junções Comunicantes/química , Humanos , Potenciais da Membrana , Oócitos , Técnicas de Patch-Clamp , Conformação Proteica , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Xenopus laevis , Proteína beta-1 de Junções ComunicantesRESUMO
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.
Assuntos
Comunicação Celular/fisiologia , Conexinas/fisiologia , Ativação do Canal Iônico/fisiologia , Animais , Permeabilidade da Membrana Celular , Conexinas/química , Humanos , Técnicas de Patch-Clamp , Fatores de Tempo , XenopusAssuntos
Conexinas/metabolismo , Canais Iônicos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Separação Celular , Conexinas/genética , Feminino , Expressão Gênica , Técnicas In Vitro , Indicadores e Reagentes , Canais Iônicos/genética , Técnicas de Patch-Clamp/instrumentação , RNA Complementar/genética , RNA Complementar/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisAssuntos
Conexinas/fisiologia , Animais , Eletrofisiologia , Oócitos/fisiologia , Relação Estrutura-Atividade , XenopusRESUMO
The effects of temperature and solvent substitution with deuterium oxide (D2O) on axoplasmic (ga) and gap junctional (gj) conductances were examined in the earthworm septate median giant axon (MGA). The temperature coefficients (Q10) for ga and gj were 1.4 and 1.5, respectively, between 5 and 15 degrees C. Substitution with D2O rapidly reduced both ga and gj by 20% and increased the Q10's to 1.5 and 1.8, respectively. The reduction in ga upon substitution with D2O and with cooling in either solvent reflects the changes that occur in solvent viscosity, which indicates that ion mobility in axoplasm, as in free solution, is primarily governed by viscous properties of the solvent. The similar initial reduction observed for gj suggests that solvent occupies the gap junction channel volume and influences transjunctional ion mobility. With time there was a further reduction in gj at 20 degrees C and a larger Q10 in D2O. The enhanced effects of D2O on gj cannot be accounted for by solvent viscosity alone and may be due to an increased hydration of the channels and/or the transport ions and by isotope effects of hydrogen-deuterium exchange on the channel protein that reduce gj.
Assuntos
Junções Intercelulares/fisiologia , Animais , Deutério , Óxido de Deutério , Condutividade Elétrica , Potenciais da Membrana , Microeletrodos , Oligoquetos , ÁguaRESUMO
Gap junction channels, now known to be formed of connexins, connect the interiors of apposed cells. These channels can be opened and closed by various physiological stimuli and experimental treatments. They are permeable to ions and neutral molecules up to a size of about 1 kDa or 1.5 nm diameter, including second messengers and metabolites. The processes of gating and of permeation are the subject of this review. Voltage is a readily applied stimulus, and transjunctional voltages, or those between cytoplasm and exterior, affect most junctions. Single channel transitions between open and closed states are rapid and presumably involve a charge movement as occurs with channels of electrically excitable channels of nerve and muscle. Identification of gating domains and charges by domain replacement and site-directed mutagenesis is being pursued. Raising cytoplasmic H+ or Ca2+ concentrations rapidly reduces junctional conductance, and this action is generally reversible, at least in part. A number of lipophilic alcohols, fatty acids and volatile anesthetics have similar actions. Phosphorylation also modulates junctional conductance, and in several cases, sites of phosphorylation are known. These gating processes appear similar to those induced by voltage. Permeability measurement indicates that the channel is aqueous and that permeation is by diffusion with only minor interactions with the channel wall. Differences among junctions are known, but further characterization of connexin and cell specificity is required.
Assuntos
Junções Intercelulares/química , Proteínas de Membrana/química , Animais , Cátions , Conexinas , Citoplasma/fisiologia , Condutividade Elétrica , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Ativação do Canal Iônico , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , PermeabilidadeRESUMO
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.
Assuntos
Conexinas/metabolismo , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Células Cultivadas , Conexina 26 , Conexinas/química , Conexinas/genética , Citoplasma/metabolismo , Junções Comunicantes/metabolismo , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus , Proteína beta-1 de Junções ComunicantesRESUMO
Steady-state and kinetic analyses of gap junctional conductance, gi, in salivary glands of Drosophila melanogaster third instar larvae reveal a strong and complex voltage dependence that can be elicited by two types of voltages. Voltages applied between the cells, i.e., transjunctional voltages, Vj, and those applied between the cytoplasm and the extracellular space, inside-outside voltages, Vi,o, markedly alter gj. Alteration of Vi-o while holding Vj = O,i.e., by equal displacement of the voltages in the cells, causes gj to increase to a maximum on hyperpolarization and to decrease to near zero on depolarization. These conductance changes associated with Vi-o are fit by a model in which there are two independent gates in series, one in each series, one in each membrane, where each gate is equally sensitive to Vi-o and exhibits first order kinetics. Vj's generated by applying voltage steps of either polarity to either cell, substantially reduce gj. These conductance changes exhibit complex kinetics that depend on Vi-o as well as Vj. At more positive Vi-o's, the changes in gj have two phases, an early phase consisting of of a decrease in gj for either polarity of Vj and a later phase consisting of an increase in gj on hyperpolarizing either cell and a decrease on depolarizing either cell. At negative Vi-o's in the plateau region of the gj-Vi-o relation, the later slow increase in gj is absent on hyperpolarizing either cell. Also, the early decrease in gj for either polarity of Vj is faster the more positive the Vi-o. The complex time course elicited by applying voltage steps to one cell can be explained as combined actions of Vi-o and Vj, with the early phase ascribable to Vj, but influenced by Vi-o, and the later phase to the changes in Vi-o associated with the generation of Vj. The substantially different kinetics and sensitivity of changes in gj by Vi-o and Vj suggests that the mechanisms of gating by these two voltages are different. Evidently, these gap-junction channels are capable of two distinct, but interactive forms of voltage dependence.
Assuntos
Junções Intercelulares/fisiologia , Animais , Drosophila melanogaster , Estimulação Elétrica , Eletrofisiologia/métodos , Larva , Matemática , Potenciais da Membrana , Microeletrodos , Glândulas Salivares/fisiologiaRESUMO
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.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Ligação Genética/genética , Mutação/genética , Cromossomo X/genética , Animais , Cálcio/farmacologia , Condutividade Elétrica , Junções Comunicantes/efeitos dos fármacos , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis , Proteína beta-1 de Junções ComunicantesRESUMO
Properties of electrotonic synapses between L14 neurons in the abdominal ganglion of the marine mollusc Aplysia californica were examined in situ and between unidentified buccal neurons maintained in tissue culture. In culture, depolarizing postsynaptic potentials in response to a train of action potentials showed apparent facilitation with increasing spike number, which was attributable to the low-pass filter properties of electrotonic transmission via gap junctions and to network properties. Gap junctional conductance (gj), calculated from current-clamp data or measured directly under voltage clamp, indicated no significant dependence of gj on transjunctional or inside-outside potential in situ or in culture. Octanol, a local anesthetic agent that reduces gj in many other systems, had no effect on gj between Aplysia neurons. The effect of intracellular acidification, a treatment that rapidly and reversibly uncouples a variety of cell types, reduced gj between Aplysia neurons but did not completely abolish it. The relationship between intracellular pH (pHi), measured with ion-sensitive microelectrodes, and gj was steeper in cultured neurons than in situ and was maximally reduced by 70-80%, as compared to 50% or less in situ at the lowest pHi values tested. The coupling coefficient (k) was reduced less by low pHi than was gj, which could be explained by a simultaneous increase in nonjunctional membrane resistance. Permeability properties of Aplysia electrotonic synapses to a variety of tracer molecules were also examined between identified L 14 neurons in situ and in dissociated buccal, abdominal, and bag neurons in culture. The fluorescent dyes Lucifer yellow, 6-carboxyfluorescein, and dichlorofluorescein (1.2-1.4 nm maximal diameters) did not spread detectably from an injected neuron to its electrically coupled neighbors, regardless of the strength of electrotonic coupling. However, the smaller tetraalkylammonium ions TMA and TEA (diameters 0.66 and 0.8 nm, concentrations measured with ion-selective electrodes), could be detected in neighboring cells within minutes. In culture, transfer of the tetraalkylammonium ions was slow and not easily detectable in cell pairs where gj was low (less than 20 nS). The permeability was as high as 10(-10) cm3/sec in situ and 10(-12) cm3/sec in culture, and values were roughly correlated with simultaneously measured values of gj. Electrotonic synapses in the nervous system of Aplysia, therefore, have a quantitatively different spectrum of sensitivities than has been found for gap junctions of other systems and appear to possess reduced permeability to tracer molecules.
Assuntos
Aplysia/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Técnicas de Cultura , Condutividade Elétrica , Eletrofisiologia , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Octanóis/farmacologia , Permeabilidade , Compostos de Amônio Quaternário/metabolismo , Tetraetilamônio , Compostos de Tetraetilamônio/metabolismoRESUMO
We have developed a procedure for the replacement of defined domains with specified domains from other proteins that we used to examine the molecular basis for the differences in voltage-dependent gating between connexins 26 (Cx26) and 32 (Cx32). This technique does not depend on sequence homology between the domains to be exchanged or the presence of restriction endonuclease sites. Rather, it makes use of a PCR strategy to create an adhesive "band-aid" that directs the annealing of the amplified sequence to the correct location in the recipient clone. With this technique we created a series of chimeras involving the replacement of topologically defined protein domains of Cx32 with the corresponding sequences of Cx26. We focused on domains that are predicted to line the gap junction channel as we expect that a component of the voltage-sensing mechanism resides there. Differences between Cx26 and Cx32 in the sequences of their first and second extracellular loops, the cytoplasmic loop, and the third transmembrane domain did not account for the difference in their calculated gating charges. Shifts along the voltage axis in the steady-state conductance-voltage relations of the chimeric connexins were produced by replacement of the first extracellular loop or the cytoplasmic loop and the amino-terminal half of the third transmembrane domain. These data suggest that the voltage-sensing mechanism arises from the interaction of domains lining the aqueous channel and domains deeper in the channel wall.