RESUMEN
Proteolytic processing of epithelial Na+ channel (ENaC) subunits has an important role in the regulation of ENaC gating. This Commentary addresses the potential roles of specific proteases and protease inhibitors in the control of Na+ channel gating.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Péptido Hidrolasas/metabolismo , Receptores de Superficie Celular/fisiología , Canales de Sodio/fisiología , Animales , Canales Epiteliales de Sodio , Femenino , Activación del Canal Iónico/fisiología , Ratones , Oocitos/fisiología , Inhibidores de Proteasas , Nexinas de Proteasas , Serina Endopeptidasas/fisiología , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , XenopusRESUMEN
We studied the cellular phosphatase inhibitors okadaic acid (OKA), calyculin A, and microcystin on the epithelial sodium channel (ENaC) in A6 renal cells. OKA increased the amiloride-sensitive current after approximately 30 min with maximal stimulation at 1-2 h. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost two times as large as in cells pretreated for 30 min with OKA, implying an increase in single channel open probability (P(o)) that doubled after OKA. Single channel analysis showed that, in cells pretreated with OKA, P(o) and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on P(o) and mean open time. An analog of OKA, okadaone, that does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks protein phosphatase 2A (PP2A) but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase that suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel.
Asunto(s)
Riñón/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Canales de Sodio/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Canales Epiteliales de Sodio , Activación del Canal Iónico/fisiología , Isomerismo , Riñón/citología , Toxinas Marinas , Microcistinas , Ácido Ocadaico/análogos & derivados , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Técnicas de Placa-Clamp/métodos , Péptidos Cíclicos/farmacología , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 2 , Canales de Sodio/químicaRESUMEN
Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.
Asunto(s)
Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular , Canales Epiteliales de Sodio , Humanos , Transporte Iónico , Potenciales de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/genética , Canales de Sodio/fisiología , XenopusRESUMEN
The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.
Asunto(s)
Células Epiteliales/metabolismo , Túbulos Renales/metabolismo , Canales de Sodio/metabolismo , Urotelio/metabolismo , Aldosterona/farmacología , Animales , Línea Celular , Polaridad Celular , Centrifugación por Gradiente de Densidad , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio , Immunoblotting , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Pruebas de Precipitina , Subunidades de Proteína , Factores de Tiempo , Urotelio/citología , Urotelio/efectos de los fármacos , Xenopus laevisRESUMEN
Na(+) absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial Na(+) channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans, controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell Na(+) currents (I(Na)) in oocytes expressing mouse alpha,beta,gamma-ENaC (mENaC) and on net Na(+) absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4-6 ml/min) with a reversible threefold stimulation of I(Na) without a change in reversal potential. The increase in I(Na) was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net Na(+) absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net Na(+) absorption, suggesting that shear stress does not influence Na(+) transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and Na(+) absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD.
Asunto(s)
Túbulos Renales Colectores/fisiología , Canales de Potasio de Rectificación Interna , Canales de Sodio/metabolismo , Amilorida/farmacología , Animales , Transporte Biológico/fisiología , Diuréticos/farmacología , Canales Epiteliales de Sodio , Expresión Génica/fisiología , Presión Hidrostática , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nefronas/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/metabolismo , Conejos , Sodio/metabolismo , Canales de Sodio/genética , Estrés Mecánico , Transfección , Xenopus laevisRESUMEN
K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca(2+)-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 +/- 1.0 to 23.4 +/- 4.7 pmol. min(-1). mm(-1) (P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl. min(-1). mm(-1). Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 +/- 1.2 vs. 9.9 +/- 2.7 pmol. min(-1). mm(-1) at 0.6 and 6 nl. min(-1). mm(-1) flow rates, respectively) or charybdotoxin (6.8 +/- 1.6 vs. 8.3 +/- 1.6 pmol. min(-1). mm(-1) at 1 and 4 nl. min(-1). mm(-1) flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.
Asunto(s)
Corteza Renal/metabolismo , Túbulos Renales Colectores/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Potasio/metabolismo , Animales , Apamina/farmacología , Western Blotting , Caribdotoxina/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Canales de Potasio de Gran Conductancia Activados por el Calcio , Mecanorreceptores/fisiología , Microscopía Fluorescente , Nefronas/efectos de los fármacos , Nefronas/metabolismo , Canales de Potasio/efectos de los fármacos , Conejos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Tetraetilamonio/farmacologíaRESUMEN
Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.
Asunto(s)
Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Canales de Sodio/química , Canales de Sodio/fisiología , Amilorida/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cisteína , Canales Epiteliales de Sodio , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Canales de Sodio/fisiología , Animales , Canales de Cloruro/fisiología , Femenino , Regulación de la Expresión Génica , Humanos , Activación del Canal Iónico , Transporte Iónico , Ratones , Oocitos , Transducción de Señal , XenopusRESUMEN
The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.
Asunto(s)
Canales de Sodio/metabolismo , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Cationes Monovalentes/metabolismo , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Canales Epiteliales de Sodio , Litio/metabolismo , Ratones , Modelos Teóricos , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp , Mutación Puntual , Potasio/metabolismo , Proteínas Recombinantes/metabolismo , Sodio/metabolismo , Canales de Sodio/genética , XenopusRESUMEN
Amiloride is a prototypic inhibitor of epithelial sodium channels. Rapid progress has been made in our understanding of the structure of the sodium channel and related cation-selective channels. This work, coupled with experiments examining how selected sodium channel mutations affect amiloride binding, provides critical clues towards defining sites within the channel that bind amiloride. Residues within the channel pore and within its extracellular domain participate in amiloride binding. These results suggest that sites that interact with amiloride within the channel's extracellular domain may be in close proximity to residues within the channel's pore.
Asunto(s)
Amilorida/farmacología , Diuréticos/farmacología , Canales de Sodio/efectos de los fármacos , Amilorida/química , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Diuréticos/química , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Riñón/efectos de los fármacos , Riñón/fisiología , Biología Molecular , Sensibilidad y Especificidad , Canales de Sodio/fisiologíaRESUMEN
Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.
Asunto(s)
Riñón/metabolismo , Canales de Sodio/metabolismo , Espectrina/metabolismo , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Línea Celular , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Regulación de la Expresión Génica/efectos de los fármacos , Riñón/citología , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Fragmentos de Péptidos/inmunología , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Canales de Sodio/química , Canales de Sodio/genética , Xenopus laevisRESUMEN
The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed alpha-, beta-, and gammaENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse alpha-, beta-, and gammaENaC (alpha-, beta-, and gammamENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length betamENaC and partial-length alpha- and gammamENaC clones were isolated. Full-length alpha- and gammamENaC cDNAs were subsequently obtained by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Injection of mouse alpha-, beta-, and gammaENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive (K(i) = 103 nM), Na(+)-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that alpha-, beta-, and gammamENaC were expressed in lung and kidney. Interestingly, alphamENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5'-RACE PCR. Its sequence was found to be nearly identical to alphamENaC. To begin to identify regions within alphamENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of alphamENaC encoding the predicted cytoplasmic NH(2) terminus. Coinjection of wild-type alpha-, beta-, and gammamENaC with the intracellular NH(2) terminus of alphamENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH(2) terminus of alphamENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.
Asunto(s)
Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Epitelio/metabolismo , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , XenopusRESUMEN
We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.
Asunto(s)
Amilorida/metabolismo , Anticuerpos Antiidiotipos/inmunología , Canales de Sodio/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/metabolismo , Secuencia de Bases , Sitios de Unión , Bovinos , Clonación Molecular , Simulación por Computador , ADN Complementario/química , Canales Epiteliales de Sodio , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
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 epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.
Asunto(s)
Canales de Sodio/metabolismo , Animales , Epitelio/metabolismo , Ratones , Mutagénesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Sodio/química , Canales de Sodio/genética , XenopusRESUMEN
Long-term exposure to cocaine can cause persistent behavioral changes and alterations in neuronal function. One cocaine-regulated mRNA in the rat brain is the beta-1 subunit of the Na+/K(+)-ATPase pump. We examined both Na+/K(+)-ATPase function and expression after cocaine treatment of pheochromocytoma cells. One-hour exposure to cocaine did not alter Na+/K(+)-ATPase activity, as measured by the ouabain-sensitive component of rubidium uptake. Four days of cocaine resulted in an approximately 30% decrease in Na+/K(+)-ATPase activity. Western blot analyses demonstrated an approximately 25% decrease in levels of the beta-1 isoform, without changes in pump total alpha subunit levels. Treatment with dopamine type 1 or type 2 receptor agonists for the same period did not affect Na+/K(+)-ATPase activity. The serotonin-selective reuptake inhibitor paroxetine caused an approximately 45% decrease in rubidium uptake after 4 days, whereas pump function was not altered after treatment with either the dopamine-selective reuptake blocker nomifensine or the norepinephrine-selective reuptake blocker desipramine. Chronic treatment with both cocaine and LY 278,584, a serotonin type 3 receptor antagonist, did not replicate the cocaine-associated decrease in pump function. Long-term cocaine exposure regulates expression and function of the Na+/K(+)-ATPase pump in neuronal-like cells; this regulation is mediated in part via the serotonin type 3 receptor. Similar Na+/K(+)-ATPase pump regulation in vivo may selectively alter neuronal function in the mammalian brain.
Asunto(s)
Cocaína/farmacología , Serotonina/fisiología , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Animales , Agonistas de Dopamina/farmacología , Células PC12 , Ratas , Receptores Adrenérgicos beta 1/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
The mammalian urinary bladder exhibits transepithelial Na+ absorption that contributes to Na+ gradients established by the kidney. Electrophysiological studies have demonstrated that electrogenic Na+ absorption across the urinary bladder is mediated in part by amiloride-sensitive Na+ channels situated within the apical membrane of the bladder epithelium. We have used a combination of in situ hybridization, Northern blot analysis, and immunocytochemistry to examine whether the recently cloned epithelial Na+ channel (ENaC) is expressed in the rat urinary bladder. In situ hybridization and Northern blot analyses indicate that alpha-, beta-, and gamma-rat ENaC (rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of alpha-, beta-, and gamma-rENaC mRNA expression in rat urinary bladder, relative to beta-actin mRNA expression, indicates that, although comparable levels of alpha- and beta-rENaC subunits are expressed in the urinary bladder of rats maintained on standard chow, the level of gamma-rENaC mRNA expression is 5- to 10-fold lower than alpha- or beta-rENaC mRNA. Immunocytochemistry, using an antibody directed against alpha-rENaC, revealed that ENaCs are predominantly localized to the luminal membrane of the bladder epithelium. Together, these data demonstrate that ENaC is expressed in the mammalian urinary bladder and suggest that amiloride-sensitive Na+ transport across the apical membrane of the mammalian urinary bladder epithelium is mediated primarily by ENaC.
Asunto(s)
Canales de Sodio/biosíntesis , Transcripción Genética , Vejiga Urinaria/metabolismo , Animales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Técnica del Anticuerpo Fluorescente Indirecta , Mamíferos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Canales de Sodio/análisis , Vejiga Urinaria/citologíaRESUMEN
The pattern of chicken intestine amiloride-binding proteins was determined using the photoreactive amiloride analogue 2'-methoxy-5'-nitrobenzamil (NMBA) and a polyclonal anti-amiloride antibody. At 10(-7)M, NMBA inhibits approximately 62% of the Na+ channel activity. At this concentration the amiloride analogue labels a number of membrane proteins, and in particular a 40-45 kDa polypeptide denoted ABP40. Incorporation of NMBA into ABP40 could be prevented by a 100-fold excess of benzamil, but not by a 1000-fold excess of 5-(N-ethyl-N-isopropyl)-amiloride. Labeling of ABP40 was intense in membranes derived from salt-deprived chickens and approximately 5-fold weaker in membranes from salt-repleted animals. Because of its small size, ABP40 is not likely to be an avian Na+ channel subunit, yet this amiloride-binding protein could be involved in the response to aldosterone.
Asunto(s)
Aldosterona/fisiología , Amilorida/metabolismo , Pollos/metabolismo , Diuréticos/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Unión ProteicaRESUMEN
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
Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.