RESUMEN
Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).
Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Concentración de Iones de Hidrógeno , Canales Iónicos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Canales de Sodio/química , Canales de Sodio/genética , Canales Iónicos Sensibles al Ácido , Animales , Quelantes/farmacología , Clonación Molecular , Canales de Sodio Degenerina , Ácido Egtácico/farmacología , Canales Epiteliales de Sodio , Cinética , Proteínas de la Membrana , Proteínas del Tejido Nervioso/metabolismo , Oocitos/metabolismo , Mutación Puntual , Unión Proteica , Canales de Sodio/metabolismo , XenopusRESUMEN
Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.
Asunto(s)
Actinas/metabolismo , Activación del Canal Iónico , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Canales Epiteliales de Sodio , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Canales de Sodio/química , Canales de Sodio/genética , Xenopus laevisRESUMEN
The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.
Asunto(s)
Actinas/química , Calcio/metabolismo , Membrana Dobles de Lípidos/química , Canales de Sodio/metabolismo , Actinas/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Calcio/química , Relación Dosis-Respuesta a Droga , Canales Epiteliales de Sodio , Cinética , Modelos Químicos , Músculos/metabolismo , Conformación Proteica , Proteolípidos/química , Conejos , Canales de Sodio/químicaRESUMEN
In search of the structural basis for gating of amiloride-sensitive Na(+) channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell-free planar lipid bilayer reconstitution system. Our results identify the N-terminus of the alpha-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy-terminal domains of beta- and gamma-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the beta- and gamma-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, "locking" the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the alpha-channel.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Canales de Sodio/química , Canales de Sodio/metabolismo , Amilorida/farmacología , Animales , Secuencia de Bases , Fenómenos Biofísicos , Biofisica , Cartilla de ADN/genética , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Cinética , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Sodio/genéticaRESUMEN
Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (alpha, beta, gamma) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of alpha-, beta-, and gamma-subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the alpha-ENaC subunit was examined in planar lipid bilayers. We report that alpha,beta,gamma-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited alpha, beta-ENaC, alpha,gamma-ENaC, and alpha-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the alpha-ENaC subunit is regulated directly by pHi.
Asunto(s)
Hidrógeno/fisiología , Membranas Intracelulares/metabolismo , Canales de Sodio/fisiología , Ácidos/farmacología , Animales , Conductividad Eléctrica , Canales Epiteliales de Sodio , Femenino , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/metabolismo , Oocitos , Ratas , Bloqueadores de los Canales de Sodio , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
Liddle's disease is an autosomal dominant form of human hypertension resulting from a basal activation of amiloride-sensitive Na+ channels (ENaC). This channel activation is produced by mutations in the beta- and/or gamma-carboxy-terminal cytoplasmic tails, in many cases causing a truncation of the last 45-76 amino acids. In this study, we tested two hypotheses; first, beta- and gamma-ENaC C-terminal truncation mutants (beta DeltaC and gamma DeltaC), in combination with the wild-type alpha-ENaC subunit, reproduce the Liddle's phenotype at the single channel level, i.e., an increase in open probability (Po), and second, these C-terminal regions of beta- and gamma-ENaC act as intrinsic blockers of this channel. Our results indicate that alpha beta DeltaC gamma DeltaC-rENaC, incorporated into planar lipid bilayers, has a significantly higher single channel Po compared to the wild-type channel (0.85 vs 0.60, respectively), and that 30-mer synthetic peptides corresponding to the C-terminal region of either beta- or gamma-ENaC block the basal-activated channel in a concentration-dependent fashion. Moreover, there was a synergy between the peptides for channel inhibition when added together. We conclude that the increase in macroscopic Na+ reabsorption that occurs in Liddle's disease is at least in part due to an increase in single channel Po and that the cytoplasmic tails of the beta- and gamma-ENaC subunits are important in the modulation of ENaC activity.
Asunto(s)
Péptidos/farmacología , Bloqueadores de los Canales de Sodio , Secuencia de Aminoácidos , Dicroismo Circular , Canales Epiteliales de Sodio , Membrana Dobles de Lípidos/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptidos/síntesis química , Canales de Sodio/metabolismo , SolucionesRESUMEN
The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.
Asunto(s)
Amilorida/farmacología , Canales de Sodio/química , Animales , Clonación Molecular , Electrofisiología , Cinética , Membrana Dobles de Lípidos/metabolismo , Unión Proteica/fisiología , Proteolípidos/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia/genética , Canales de Sodio/genética , XenopusRESUMEN
The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.
Asunto(s)
Canales de Sodio/metabolismo , Aldosterona/farmacología , Secuencia de Aminoácidos , Anticuerpos/inmunología , Línea Celular , Electroforesis en Gel de Poliacrilamida , Membrana Dobles de Lípidos , Metilación , Canales de Sodio/inmunologíaRESUMEN
We reported the identification of three outwardly rectified Cl- channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of anion- and cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 microg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 +/- 9 pS in symmetrical Cl- solutions, halide selectivity sequence of I- > Cl- > Br-, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-Galphai antibodies to precipitate Galphai protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Galphai protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.
Asunto(s)
Canales de Calcio/fisiología , Células Epiteliales/fisiología , Tráquea/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Anticuerpos , Western Blotting , Canales de Calcio/biosíntesis , Canales de Calcio/aislamiento & purificación , Bovinos , Cloruros/metabolismo , Cromatografía por Intercambio Iónico , Conductividad Eléctrica , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/citología , Proteínas de Unión al GTP/metabolismo , Membrana Dobles de Lípidos , Sustancias Macromoleculares , Potenciales de la Membrana , Peso Molecular , Tráquea/citologíaRESUMEN
Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpharENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant alpharENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type alpharENaC was inhibited by amiloride with a Ki of 169 nM. Deletion of the entire WYRFHY tract (alpharENaC Delta278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (Ki = 26.5 microM). Similar results were obtained when either alpharENaC or alpharENaC Delta278-283 were co-expressed with wt beta- and gammarENaC (Ki values of 155 nM and 22.8 microM, respectively). Moreover, alpharENaC H282D was insensitive to submicromolar concentrations of amiloride (Ki = 6.52 microM), whereas alpharENaC H282R was inhibited by amiloride with a Ki of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpharENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gammarENaC alters amiloride's Ki, suggest that selected regions of the extracellular loop of alpharENaC may be in close proximity to residues within the channel pore.
Asunto(s)
Amilorida/química , Canales de Sodio/química , Actinas/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Conductividad Eléctrica , Epitelio , Histidina/química , Técnicas Inmunológicas , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos , Potenciales de la Membrana , Oocitos , Técnicas de Placa-Clamp , Proteínas Recombinantes , Eliminación de Secuencia , Bloqueadores de los Canales de Sodio , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.
Asunto(s)
Actinas/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Sistema Respiratorio/metabolismo , Canales de Sodio/metabolismo , Actinas/farmacología , Adenosina Trifosfato/farmacología , Animales , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Epitelio/metabolismo , Gelsolina/farmacología , Isomerismo , Membrana Dobles de Lípidos/metabolismo , Conejos , Ratas , Canales de Sodio/efectos de los fármacosRESUMEN
Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/fisiología , Alveolos Pulmonares/metabolismo , Canales de Sodio/metabolismo , Amilorida/farmacología , Animales , Bovinos , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Células Epiteliales , Epitelio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Masculino , Toxina del Pertussis , Fosforilación , Alveolos Pulmonares/citología , Conejos , Canales de Sodio/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.
Asunto(s)
Amilorida/farmacología , Activación del Canal Iónico/fisiología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Diuréticos/farmacología , Electrofisiología , Canales Epiteliales de Sodio , Expresión Génica , Activación del Canal Iónico/efectos de los fármacos , Cinética , Membrana Dobles de Lípidos , Liposomas/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Mutación Puntual , Potasio/metabolismo , Canales de Sodio/química , XenopusRESUMEN
A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+.
Asunto(s)
Calcio/farmacología , Membrana Dobles de Lípidos , Canales de Sodio/fisiología , Amilorida/farmacología , Animales , Perros , Conductividad Eléctrica , Epitelio/fisiología , Activación del Canal Iónico , Cinética , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Microsomas/metabolismo , Modelos Químicos , Técnicas de Placa-Clamp , Biosíntesis de Proteínas , Conejos , Reticulocitos/metabolismo , Canales de Sodio/biosíntesis , Canales de Sodio/químicaRESUMEN
We have recently cloned the alpha subunit of a bovine amiloride-sensitive Na+ channel (alphabENaC). This subunit shares extensive homology with both rat and human alphaENaC subunits but shows marked divergence at the C terminus beginning at amino acid 584 of the 697-residue sequence. When incorporated into planar lipid bilayers, alphabENaC almost exclusively exhibits a main transition to 39 picosiemens (pS) with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). In contrast, the alpha subunit of the rat renal homolog of ENaC (alpharENaC) has a main transition step to 13 pS that is almost constituitively open, with a second stepwise transition of 26 to 39 pS. A deletion mutant of alphabENaC, encompassing the entire C-terminal region (R567X), converts the kinetic behavior of alphabENaC to that of alpharENaC, i. e. a transition to 13 pS followed by a second 26 pS transition to 39 pS. Chemical cross-linking of R567X restores the wild-type alphabENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produced only 13 pS transitions. In contrast, an equivalent C-terminal truncation of alpharENaC (R613X) had no effect on the gating pattern of alpharENaC. These results are consistent with the hypothesis that interactions between the C termini of alphabENaC account for the different kinetic behavior of this member of the ENaC family of Na+ channels.
Asunto(s)
Amilorida/farmacología , Canales de Sodio/química , Secuencia de Aminoácidos , Animales , Bovinos , Reactivos de Enlaces Cruzados , ADN Complementario , Humanos , Activación del Canal Iónico , Cinética , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Concentración Osmolar , Ratas , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Cloruro de Sodio , Reactivos de Sulfhidrilo , XenopusRESUMEN
Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)P4) has a direct biphasic (activation/inhibition) effect on an epithelial Ca(2+)-activated chloride channel. The effect of D-Ins(3,4,5,6)P4 is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)P4 on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an "orphan" messenger. They suggest that the molecular target for D-Ins(3,4,5,6)P4 is a plasma membrane Ca(2+)-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)P4 and Ca2+ may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis.
Asunto(s)
Calcio/farmacología , Canales de Cloruro/fisiología , Fosfatos de Inositol/farmacología , Adenosina Trifosfato/farmacología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/farmacología , Calmodulina/farmacología , Bovinos , Canales de Cloruro/biosíntesis , Canales de Cloruro/efectos de los fármacos , Clonación Molecular , Ácido Egtácico/farmacología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Membrana Dobles de Lípidos , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transducción de Señal , Xenopus laevisRESUMEN
Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Activación del Canal Iónico , Canales de Sodio/metabolismo , Actinas/farmacología , Adenosina Trifosfato/farmacología , Animales , Epitelio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genéticaRESUMEN
We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.
Asunto(s)
Proteínas Quinasas/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Colforsina/farmacología , Epitelio/efectos de los fármacos , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , XenopusRESUMEN
Amiloride-sensitive Na+ channels play a vital role in many important physiological processes such as delineation of the final urine composition, sensory transduction, and whole-body Na+ homeostasis. These channels display a wide range of biophysical properties, and are regulated by cAMP-mediated second messenger systems. The first of these channels has recently been cloned. This cloned amiloride-sensitive Na+ channel is termined ENaC (Epithelial Na+ Channel) and, in heterologous cellular expression systems, displays a single channel conductance of 4 to 7 pS, a high PNa/PK (> 10), a high amiloride sensitivity (Ki(amil) = 150 nM), and relatively long open and closed times. ENaC may form the core conduction element of many of these functionally diverse forms of Na+ channel. The kinetic and regulatory differences between these channels may be due, in large measure, to unique polypeptides that associate with the core element, forming a functional channel unit.
Asunto(s)
Amilorida/farmacología , Diuréticos/farmacología , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología , Animales , Humanos , Canales de Sodio/químicaRESUMEN
Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.