RESUMEN
Acid sensing ion channel 3 (ASIC3) is a cation channel gated by extracellular protons. It is highly expressed in sensory neurons, including small nociceptive neurons and has been proposed to participate in pain perception associated with tissue acidosis and in mechanoperception. Neuropeptide FF (NPFF) and FMRFamide have been shown to potentiate proton-gated currents from cultured sensory neurons and acid sensing ion channel (ASIC) cDNA transfected cells. In this study, we report that another mammalian peptide neuropeptide SF (NPSF), derived from the same precursor, also considerably increases the amplitude of the sustained current of heterologously expressed ASIC3 (12-fold vs. 19- and nine-fold for FMRFamide and NPFF, respectively) with an EC(50) of approximately 50 microM. Similar effects were also observed on endogenous ASIC3-like sustained current recorded from DRG neurons although of smaller amplitudes (two-, three- and seven-fold increase for NPSF, NPFF and FMRFamide, respectively), and essentially related to a slowing down of the inactivation rate. Importantly, this modulation induced changes in neuronal excitability in response to an electrical stimulus applied during extracellular acidification. ASIC3-mediated sustained depolarisation, and its regulation by neuropeptides, could thus be important in regulating polymodal neuron excitability particularly under inflammatory conditions where the expression levels of both NPFF precursor and ASIC3 are increased.
Asunto(s)
Proteínas de la Membrana , Proteínas del Tejido Nervioso , Neuronas Aferentes/efectos de los fármacos , Neuropéptidos/farmacología , Canales de Sodio/fisiología , Canales Iónicos Sensibles al Ácido , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Neuronas Aferentes/fisiología , Ratas , Ratas WistarRESUMEN
The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.
Asunto(s)
FMRFamida/farmacología , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Canales de Sodio/química , Sodio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Cisteína/química , ADN Complementario/genética , Femenino , Humanos , Mesilatos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Oocitos , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Electricidad Estática , Reactivos de Sulfhidrilo/farmacología , Xenopus laevisRESUMEN
Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in sensory neurons, where they are thought to be involved in pain perception associated with tissue acidosis. They are also expressed in brain. A number of brain regions, like the hippocampus, contain large amounts of chelatable vesicular Zn(2+). This paper shows that Zn(2+) potentiates the acid activation of homomeric and heteromeric ASIC2a-containing channels (i.e. ASIC2a, ASIC1a+2a, ASIC2a+3), but not of homomeric ASIC1a and ASIC3. The EC(50) for Zn(2+) potentiation is 120 and 111 microm for the ASIC2a and ASIC1a+2a current, respectively. Zn(2+) shifts the pH dependence of activation of the ASIC1a+2a current from a pH(0.5) of 5.5 to 6.0. Systematic mutagenesis of the 10 extracellular histidines of ASIC2a leads to the identification of two residues (His-162 and His-339) that are essential for the Zn(2+) potentiating effect. Mutation of another histidine residue, His-72, abolishes the pH sensitivity of ASIC2a. This residue, which is located just after the first transmembrane domain, seems to be an essential component of the extracellular pH sensor of ASIC2a.
Asunto(s)
Ácidos/metabolismo , Histidina/metabolismo , Hidrógeno/metabolismo , Canales Iónicos/metabolismo , Zinc/metabolismo , Animales , Cationes , Femenino , Concentración de Iones de Hidrógeno , Canales Iónicos/química , Canales Iónicos/genética , Mutagénesis Sitio-Dirigida , Xenopus laevisRESUMEN
Amiloride-sensitive Na(+) channels belonging to the recently discovered NaC/DEG family of genes have been found in several human tissues including epithelia and central and peripheral neurons. We describe here the molecular cloning of a cDNA encoding a novel human amiloride-sensitive Na(+) channel subunit that is principally expressed in the small intestine and has been called hINaC (human intestine Na(+) channel). This protein is similar to the recently identified rodent channel BLINaC and is relatively close to the acid sensing ion channels (ASICs) (79 and 29% amino acid identity, respectively). ASICs are activated by extracellular protons and probably participate in sensory neurons to nociception linked to tissue acidosis. hINaC is not activated by lowering the external pH but gain-of-function mutations can be introduced and reveal when expressed in Xenopus oocytes, an important Na(+) channel activity which is blocked by amiloride (IC(50)=0.5 microM). These results suggest the existence of a still unknown physiological activator for hINaC (e.g. an extracellular ligand). The presence of this new amiloride-sensitive Na(+) channel in human small intestine probably has interesting physiological as well as physiopathological implications that remain to be clarified. The large activation of this channel by point mutations may be associated with a degenerin-like behavior as previously observed for channels expressed in nematode mechanosensitive neurons. The hINaC gene has been mapped on the 4q31.3-q32 region of the human genome.
Asunto(s)
Amilorida/farmacología , Intestino Delgado/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Mapeo Físico de Cromosoma , Bloqueadores de los Canales de Sodio , Canales de Sodio/genética , Canales Iónicos Sensibles al Ácido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 4/genética , Clonación Molecular , Canales de Sodio Degenerina , Conductividad Eléctrica , Canales Epiteliales de Sodio , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Canales Iónicos/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Oocitos , Especificidad de Órganos , Filogenia , Mutación Puntual/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
1. A degenerate polymerase chain reaction (PCR) homology screening procedure was applied to rat brain cDNA in order to identify novel genes belonging to the amiloride-sensitive Na+ channel and degenerin (NaC/DEG) family of ion channels. A single gene was identified that encodes a protein related to but clearly different from the already cloned members of the family (18-30 % amino acid sequence identity). Phylogenetic analysis linked this protein to the group of ligand-gated channels that includes the mammalian acid-sensing ion channels and the Phe-Met-Arg-Phe-amide (FMRFamide)-activated Na+ channel. 2. Expression of gain-of-function mutants after cRNA injection into Xenopus laevis oocytes or transient transfection of COS cells induced large constitutive currents. The activated channel was amiloride sensitive (IC50, 1.31 microM) and displayed a low conductance (9-10 pS) and a high selectivity for Na+ over K+ (ratio of the respective permeabilities, PNa+/PK+ >= 10), all of which are characteristic of NaC/DEG channel behaviour. 3. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed a predominant expression of its mRNA in the small intestine, the liver (including hepatocytes) and the brain. This channel has been called the brain-liver-intestine amiloride-sensitive Na+ channel (BLINaC). 4. Corresponding gain-of-function mutations in Caenorhabditis elegans degenerins are responsible for inherited neurodegeneration in the nematode. Besides the BLINaC physiological function that remains to be established, mutations in this novel mammalian degenerin-like channel might be of pathophysiological importance in inherited neurodegeneration and liver or intestinal pathologies.
Asunto(s)
Canales Iónicos/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Canales de Sodio/biosíntesis , Canales de Sodio/genética , Canales Iónicos Sensibles al Ácido , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Biofisica , Northern Blotting , Células COS , Mapeo Cromosómico , Clonación Molecular , Canales de Sodio Degenerina , Canales Epiteliales de Sodio , Hibridación Fluorescente in Situ , Canales Iónicos/genética , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , XenopusRESUMEN
H(+)-gated cation channels are members of a new family of ionic channels, which includes the epithelial Na+ channel and the FMRFamide-activated Na+ channel. ASIC, the first member of the H(+)-gated Na+ channel subfamily, is expressed in brain and dorsal root ganglion cells (DRGs). It is activated by pHe variations below pH 7. The presence of this channel throughout the brain suggests that the H+ might play an essential role as a neurotransmitter or neuromodulator. The ASIC channel is also present in dorsal root ganglion cells, as is its homolog DRASIC, which is specifically present in DRGs and absent in the brain. Since external acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer, these two channel proteins are potentially central players in pain perception. ASIC activates and inactivates rapidly, while DRASIC has both a fast and sustained component. Other members of this family such as MDEG1 and MDEG2 are either H(+)-gated Na+ channels by themselves (MDEG1) or modulators of H(+)-gated channels formed by ASIC and DRASIC. MDEG1 is of particular interest because the same mutations that produce selective neurodegeneration in C. elegans mechanosensitive neurons, when introduced in MDEG1, also produce neurodegeneration. MDEG2 is selectively expressed in DRGs, where it assembles with DRASIC to radically change its biophysical properties, making it similar to the native H(+)-gated channel, which is presently the best candidate for pain perception.
Asunto(s)
Encéfalo/metabolismo , Proteínas de la Membrana , Canales de Sodio/metabolismo , Canales Iónicos Sensibles al Ácido , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Canales de Sodio Degenerina , Canales Epiteliales de Sodio , Ganglios Espinales/metabolismo , Hibridación in Situ , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/metabolismo , Alineación de SecuenciaRESUMEN
The acid-sensing ion channel (ASIC) subunits ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive Na+ channel/degenerin family of ion channels. They form proton-gated channels that are expressed in the central nervous system and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2, ASIC2b, is not active on its own but modifies the properties of ASIC3. In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for Na+ over K+, ASIC3/ASIC2b heteromultimers show a nonselective component. Chimeras of the two splice variants allowed identification of a 9-amino acid region preceding the first transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion permeation and is critical for Na+ selectivity. Three amino acids in this region (Ile-19, Phe-20, and Thr-25) appear to be particularly important, because channels mutated at these residues discriminate poorly between Na+ and K+. In addition, the pH dependences of the activity of the F20S and T25K mutants are changed as compared with that of wild-type ASIC2. A corresponding ASIC3 mutant (T26K) also has modified Na+ selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore.
Asunto(s)
Canales Iónicos/química , Proteínas del Tejido Nervioso/química , Canales de Potasio/química , Canales de Sodio/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células COS , Canales de Sodio Degenerina , Electrofisiología , Canales Epiteliales de Sodio , Canales Iónicos/genética , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismoRESUMEN
Amiloride sensitivity is a common characteristic of structurally related cationic channels that are associated with a wide range of physiological functions. In Caenorhabditis elegans, neuronal and muscular degenerins are involved in mechanoperception. In animal epithelia, a Na(+)-selective channel participates in vectorial Na+ transport. In the snail nervous system, an ionotropic receptor for the peptide FMRFamide forms a Na(+)-selective channel. In mammalian brain and/or in sensory neurons, ASIC channels form H(+)-activated cation channels involved in nociception linked to acidosis. We have now cloned a new member of this family from Drosophila melanogaster. The corresponding protein displays low sequence identity with the previously cloned members of the super-family but it has the same structural organization. Its mRNA was detected from late embryogenesis (14-17 hours) and was present in the dendritic arbor subtype of the Drosophila peripheral nervous system multiple dendritic (md) sensory neurons. While the origin and specification of md neurons are well documented, their roles are still poorly understood. They could function as stretch or touch receptors, raising the possibility that this Drosophila gene product, called dmdNaC1, could also be involved in mechanotransduction.
Asunto(s)
Amilorida/farmacología , Drosophila melanogaster/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Datos de Secuencia Molecular , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/embriología , Nervios Periféricos/metabolismo , Reacción en Cadena de la Polimerasa , Canales de Sodio/genéticaRESUMEN
The Helix aspersa Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channel is formed by homomultimerization of several FMRFamide-activated Na+ channel (FaNaCh) proteins. FaNaCh is homologous to the subunits that compose the amiloride-sensitive epithelial sodium channel, to Caenorhabditis elegans degenerins, and to acid-sensing ionic channels. FaNaCh properties were analyzed in stably transfected human embryonic kidney cells (HEK-293). The channel was functional with an EC50 for FMRFamide of 1 microM and an IC50 (25 degreesC) for amiloride of 6.5 microM as assessed by 22Na+ uptake measurements. The channel activity was associated with the presence of a protein at the cell surface with an apparent molecular mass of 82 kDa. The 82-kDa form was derived from an incompletely glycosylated form of 74 kDa found in the endoplasmic reticulum. Formation of covalent bonds between subunits of the same complex were observed either after formation of intersubunit disulfide bonds following cell homogenization and solubilization with Triton X-100 or after use of bifunctional cross-linkers. This resulted in the formation of covalent multimers that contained up to four subunits. Hydrodynamic properties of the solubilized FaNaCh complex also indicated a maximal stoichiometry of four subunits per complex. It is likely that epithelial Na+ channels, acid-sensing ionic channels, degenerins, and the other proteins belonging to the same ion channel superfamily also associate within tetrameric complexes.
Asunto(s)
Conformación Proteica , Canales de Sodio/química , Animales , Caenorhabditis elegans , Línea Celular , Dimerización , Caracoles Helix , Humanos , Canales de Sodio/genética , Canales de Sodio/metabolismo , Relación Estructura-Actividad , Transfección , XenopusRESUMEN
Amiloride-sensitive sodium channels have been implicated in reproductive and early developmental processes of several species. These include the fast block of polyspermy in Xenopus oocytes that follows the sperm binding to the egg or blastocoel expansion in mammalian embryo. We have now identified a gene called dGNaC1 that is specifically expressed in the gonads and early embryo in Drosophila melanogaster. The corresponding protein belongs to the superfamily of cationic channels blocked by amiloride that includes Caenorhabditis elegans degenerins, the Helix aspersa FMRF-amide ionotropic receptor (FaNaC), the mammalian epithelial Na+ channel (ENaC), and acid-sensing ionic channels (ASIC, DRASIC, and MDEG). Expression of dGNaC1 in Xenopus oocytes generates a constitutive current that does not discriminate between Na+ and Li+, but is selective for Na+ over K+. This current is blocked by amiloride (IC50 = 24 microM), benzamil (IC50 = 2 microM), and ethylisopropyl amiloride (IC50 = 49 microM). These properties are clearly different from those obtained after expression of the previously cloned members of this family, including ENaC and the human alphaENaC-like subunit, deltaNaC. Interestingly, the pharmacology of dGNaC1 is not very different from that found for the Na+ channel characterized in rabbit preimplantation embryos. We postulate that this channel may participate in gametogenesis and early embryonic development in Drosophila.
Asunto(s)
Canales de Sodio/biosíntesis , Canales de Sodio/fisiología , Testículo/metabolismo , Amilorida/análogos & derivados , Amilorida/metabolismo , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Cationes/metabolismo , Proteínas de Drosophila , Drosophila melanogaster , Embrión no Mamífero/fisiología , Canales Epiteliales de Sodio , Femenino , Caracoles Helix , Humanos , Masculino , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Oocitos/fisiología , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Caracteres Sexuales , Sodio/metabolismo , Canales de Sodio/química , Canales de Sodio/genética , Interacciones Espermatozoide-Óvulo , Xenopus laevisRESUMEN
MDEG1 is a cation channel expressed in brain that belongs to the degenerin/epithelial Na+ channel superfamily. It is activated by the same mutations which cause neurodegeneration in Caenorhabditis elegans if present in the degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence identity with the recently cloned proton-gated cation channel ASIC (acid sensing ion channel), a new member of the family which is present in brain and in sensory neurons. We have now identified MDEG1 as a proton-gated channel with properties different from those of ASIC. MDEG1 requires more acidic pH values for activation and has slower inactivation kinetics. In addition, we have cloned from mouse and rat brain a splice variant form of the MDEG1 channel which differs in the first 236 amino acids, including the first transmembrane region. This new membrane protein, which has been called MDEG2, is expressed in both brain and sensory neurons. MDEG2 is activated neither by mutations that bring neurodegeneration once introduced in C. elegans degenerins nor by low pH. However, it can associate both with MDEG1 and another recently cloned H+-activated channel DRASIC to form heteropolymers which display different kinetics, pH dependences, and ion selectivities. Of particular interest is the subunit combination specific for sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it gives rise to a biphasic current with a sustained current which discriminates poorly between Na+ and K+, like the native H+-gated current recorded in dorsal root ganglion cells. This sustained current is thought to be required for the tonic sensation of pain caused by acids.
Asunto(s)
Encéfalo/metabolismo , Ganglios Espinales/metabolismo , Canales Iónicos/química , Canales Iónicos/metabolismo , Proteínas de la Membrana , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio/metabolismo , Canales de Sodio/química , Canales de Sodio/metabolismo , Canales Iónicos Sensibles al Ácido , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , Canales de Sodio Degenerina , Canales Epiteliales de Sodio , Canales Iónicos/genética , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Canales de Potasio/química , Canales de Potasio/genética , ARN Mensajero/metabolismo , Ratas , Canales de Sodio/genética , Distribución TisularRESUMEN
Three homologous subunits of the amiloride-sensitive Na+ channel, entitled alpha, beta, and gamma, have been cloned either from distal colon of a steroid-treated rat or from human lung. The alpha, beta, and gamma subunits have similarities with degenerins, a family of proteins found in the mechanosensory neurons of the nematode Caenorhabditis elegans. All these proteins are characterized by the presence of a large extracellular domain, located between two transmembrane alpha-helices, and by short NH2 and COOH terminal cytoplasmic segments. They constitute the first members of a new gene super-family of ionic channels. The epithelial Na+ channel is specifically expressed at the apical membrane of Na(+)-reabsorbing epithelial cells. Its activity is controlled by several distinct hormones, especially by corticosteroids. These hormones act either transcriptionally (such as aldosterone in distal colon, or glucocorticoids in lung) and/or post-transcriptionally (such as aldosterone in kidney). Recent works have provided new insights in the function of that important osmoregulatory system.
Asunto(s)
Amilorida/farmacología , Fragmentos de Péptidos/genética , ARN Mensajero/análisis , Canales de Sodio/efectos de los fármacos , Esteroides/farmacología , Animales , Epitelio/química , Epitelio/efectos de los fármacos , Humanos , Familia de Multigenes , Relación Estructura-ActividadRESUMEN
The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits, alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5 pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha-, beta-, and gamma-proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-NH2 peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic and contain potential regulatory segments that are able to modulate the activity of the channel. Accordingly, in Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta- and gamma-chains of the epithelial Na+ channel lead to a hyperactivity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase in beta- and gamma-subunit transcription is observed, whereas the alpha-subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.
Asunto(s)
Amilorida/farmacología , Epitelio/fisiología , Canales de Sodio/fisiología , Animales , Fibrosis Quística/etiología , ADN Complementario , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , ARN Mensajero , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Dominios Homologos src/fisiologíaRESUMEN
The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha, beta, and gamma proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-amide peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic, and contain potential regulatory segments that are able to modulate the activity of the channel. In Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta and gamma chains of the epithelial Na+ channel lead to a hyper-activity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting, by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase of the beta and gamma subunits transcription is observed, whereas the alpha subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not significantly altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are the positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.
Asunto(s)
Amilorida/farmacología , Canales de Sodio , Animales , Humanos , Cinética , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Canales de Sodio/fisiologíaRESUMEN
The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and structurally related peptides are present both in invertebrate and vertebrate nervous systems. Although they constitute a major class of invertebrate peptide neurotransmitters, the molecular structure of their receptors has not yet been identified. In neurons of the snail Helix aspersa, as well as in Aplysia bursting and motor neurons, FMRFamide induces a fast excitatory depolarizing response due to direct activation of an amiloride-sensitive Na+ channel. We have now isolated a complementary DNA from Helix nervous tissue; when expressed in Xenopus oocytes, it encodes an FMRFamide-activated Na+ channel (FaNaCh) that can be blocked by amiloride. The corresponding protein shares a very low sequence identity with the previously cloned epithelial Na+ channel subunits and Caenorhabditis elegans degenerins, but it displays the same overall structural organization. To our knowledge, this is the first characterization of a peptide-gated ionotropic receptor.
Asunto(s)
Canales de Sodio/genética , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans , Células Cultivadas , Clonación Molecular , ADN Complementario , FMRFamida , Caracoles Helix , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Datos de Secuencia Molecular , Neuropéptidos/fisiología , Ratas , Proteínas Recombinantes/farmacología , Bloqueadores de los Canales de Sodio , XenopusRESUMEN
Molecular cloning of the amiloride-sensitive Na+ channel has permitted analysis of the mechanisms of its stimulation by steroids. In rat lung cells in primary culture, where its mRNA has been detected, the activity of an amiloride-sensitive channel, highly selective for Na+, is controlled by corticosteroids. Dexamethasone (0.1 microM) or aldosterone (1 microM) induced, after a minimum 10 h treatment, a large increase of the amiloride-induced hyperpolarization and of the amiloride-sensitive current. A parallel increase in the amount of the mRNA was observed. The corresponding gene is thus a target for steroid action. Using synthetic specific agonists and antagonists for mineralo- and glucocorticoid receptors, it has been shown that the steroid action on Na+ channel expression is mediated via glucocorticoid receptors. Triiodothyronine, known to modulate steroid action in several tissues, had no effect on both the amiloride-sensitive Na+ current and the level of the mRNA for the Na+ channel protein, but potentiates the stimulatory effect of dexamethasone. The increase in Na+ channel activity observed in the lung around birth can thus be explained by a direct increase in transcription of the Na+ channel gene.
Asunto(s)
Aldosterona/farmacología , Amilorida/farmacología , Dexametasona/farmacología , Regulación de la Expresión Génica , Pulmón/metabolismo , Canales de Sodio/genética , Animales , Northern Blotting , Células Cultivadas , Clonación Molecular , Pulmón/citología , Pulmón/efectos de los fármacos , Antagonistas de Receptores de Mineralocorticoides , Ratas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Canales de Sodio/metabolismoRESUMEN
Long term regulation of the amiloride-sensitive Na+ channel activity by steroid hormones occurs via de novo protein synthesis. The messenger level of RCNaCh1, previously shown by expression cloning to be a component of this channel, was measured in colons from rats fed with a low sodium diet. After 1 week of this diet, the channel activity was increased in an all-or-none fashion, whereas the level of RCNaCh1 messenger remained constant. A cDNA coding for another subunit of the Na+ channel was obtained by polymerase chain reaction. The 650-amino acid protein, entitled RCNaCh2, is 58% homologous to RCNaCh1 and displays a similar structure. It had no intrinsic activity when expressed alone in Xenopus oocytes, but its co-expression with RCNaCh1 increased the channel activity 18 +/- 5-fold. The increase in messenger level for RCNaCh2 during the time course of the diet is likely to explain the positive regulation of the rat colon Na+ channel by steroids. Immunocytochemical localization of the RCNaCh1 subunit revealed an apical labeling in colon from sodium-depleted rats. No labeling was observed in colon from control animals. These results suggest that oligomerization is needed for the proper expression of RCNaCh1 at the cell surface.
Asunto(s)
Aldosterona/fisiología , Amilorida/farmacología , Canales de Sodio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Regulación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , XenopusRESUMEN
A key protein component of the amiloride-sensitive sodium channel has been cloned from rat colon and human lung. It may represent the first member of a new family of ionic channels expressed from nematode to human. The biochemical properties of the rat protein, a 699 amino acids long polypeptide, have been analyzed. Four polyclonal antibodies raised against distinct parts of the channel immunoprecipitated a glycosylated protein of 96 kDa after cRNA expression in oocytes as well as after in vitro translation. When expressed alone into oocytes, the protein was not stable; most of it remains stacked into the endoplasmic reticulum. This results in a very low yield of complete maturation of the protein at the cell surface after expression from the pure cRNA. To determine the membrane topology of the protein, in vitro translation by a rabbit reticulocyte lysate was performed followed by insertion into canine pancreatic microsomes and protease digestion. Analysis revealed a model with only two transmembrane alpha helices and a large extracellular domain of about 500 amino acids. The NH2 and COOH termini are cytoplasmic. Protease digestion results suggest the possible presence of a structural element that could have a function similar to that of the H5 segment in K+ channels. The model indicates that there is no cytoplasmic site for protein kinase A phosphorylation. The well known regulation of the channel activity by hormones that activate this kinase such as vasopressin might thus be situated on another channel component.
Asunto(s)
Amilorida/farmacología , Canales de Sodio/química , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Canales de Sodio/efectos de los fármacos , XenopusRESUMEN
Water balance in the lung is controlled via active Na+ and Cl- transport. Electrophysiological measurements on lung epithelial cells demonstrated the presence of a Na+ channel that is inhibited by amiloride (K0.5 = 90 nM) and some of its derivatives such as phenamil (K0.5 = 19 nM) and benzamil (K0.5 = 14 nM) but not by ethylisopropylamiloride. An amiloride-sensitive Na+ channel of 4 pS was recorded from outside-out patches excised from the apical membrane. This channel is highly selective for Na+ (PNa+/PK+ > or = to 10). Isolation of a human lung cDNA led to the primary structure of the lung Na+ channel. The corresponding protein is 669 residues long and has two large hydrophobic domains. An amiloride-sensitive Na(+)-selective current apparently identical to the one observed in lung epithelial cells was recorded after expression of the cloned channel in oocytes. The level of the mRNA for the Na+ channel was highly increased from fetal to newborn and adult stages. This observation indicates that the increased Na+ reabsorption that occurs at birth as a necessary event to pass to an air-breathing environment is probably associated with control of transcription of this Na+ channel. The human gene for the lung Na+ channel was mapped on chromosome 12p13.