RESUMEN
The second FMRFamide-gated Na(+) channel (HtFaNaC), from Helisoma trivolvis, has been cloned. HtFaNaC has some different pharmacological properties to HaFaNaC, from Helix aspersa, which has enabled a rational approach to be made to start to identify the FMRFamide recognition site. Several chimeras were made by switching sections between the channels. The differences in sensitivity to FMRFamide, and amiloride, were assessed after expression in Xenopus oocytes. The data suggest that a recognition site for FMRFamide, and the potentiating action of amiloride, resides in a sequence of about 120 amino acids in the extracellular loop proximal to the first transmembrane segment.
Asunto(s)
FMRFamida/farmacología , Moluscos/metabolismo , Canales de Sodio/fisiología , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Diuréticos/farmacología , Electrofisiología , Datos de Secuencia Molecular , Oocitos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/fisiología , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Transfección , Xenopus laevisRESUMEN
We examined the effects of calcium channel beta subunits upon the recovery from inactivation of alpha(1) subunits expressed in Xenopus oocytes. Recovery of the current carried by the L-type alpha(1) subunit (cyCa(v)1) from the jellyfish Cyanea capillata was accelerated by coexpression of any beta subunit, but the degree of potentiation differed according to which beta isoform was coexpressed. The Cyanea beta subunit was most effective, followed by the mammalian b(3), b(4), and beta(2a) subtypes. Recovery of the human Ca(v)2.3 subunit was also modulated by beta subunits, but was slowed instead. beta(3) was the most potent subunit tested, followed by beta(4), then beta(2a), which had virtually no effect. These results demonstrate that different beta subunit isoforms can affect recovery of the channel to varying degrees, and provide an additional mechanism by which beta subunits can differentially regulate alpha(1) subunits.
Asunto(s)
Canales de Calcio/fisiología , Animales , Canales de Calcio/genética , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Estimulación Eléctrica , Femenino , Regulación de la Expresión Génica , Humanos , Potenciales de la Membrana/fisiología , Oocitos , Subunidades de Proteína , ARN/administración & dosificación , ARN/genética , Escifozoos , XenopusRESUMEN
We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH(2) (FMRFamide)-gated Na(+) channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC(50) = 70 microM) than HaFaNaC (EC(50) = 2 microM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na(+) channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.
Asunto(s)
Amilorida/análogos & derivados , FMRFamida/metabolismo , Activación del Canal Iónico/genética , Neuronas/metabolismo , Caracoles/genética , Canales de Sodio/genética , Amilorida/farmacología , Animales , Células Cultivadas , Clonación Molecular , FMRFamida/farmacología , Expresión Génica , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Homología de Secuencia de Aminoácido , Caracoles/metabolismo , Canales de Sodio/biosíntesis , Canales de Sodio/efectos de los fármacos , Transfección , XenopusRESUMEN
The importance of voltage-gated Ca(2+) channels in cellular function is illustrated by the many distinct types of Ca(2+) currents found in vertebrate tissues, a variety that is generated in part by numerous genes encoding Ca(2+) channel subunits. The degree to which this genetic diversity is shared by invertebrates has only recently become apparent. Cloning of Ca(2+) channel subunits from various invertebrate species, combined with the wealth of information from the Caenorhabditis elegans genome, has clarified the organization and evolution of metazoan Ca(2+) channel genes. Functional studies have employed novel structural information gained from invertebrate Ca(2+) channels to complement ongoing research on mammalian Ca(2+) currents, while demonstrating that the strict correspondence between pharmacological and molecular classes of vertebrate Ca(2+) channels does not fully extend to invertebrate tissues. Molecular structures can now be combined with physiological data to develop a more cogent system of categorizing invertebrate channel subtypes. In this review, we examine recent progress in the characterization of invertebrate Ca(2+) channel genes and its relevance to the diversity of invertebrate Ca(2+) currents.
Asunto(s)
Canales de Calcio/genética , Invertebrados/genética , Activación del Canal Iónico , Animales , Evolución Biológica , Caenorhabditis elegans/genética , Canales de Calcio/fisiología , Clonación Molecular , Conductividad Eléctrica , Humanos , Invertebrados/fisiologíaRESUMEN
In high voltage-activated calcium channels, the binding between the pore-forming alpha1 subunit and the modulatory beta subunit is mediated by interaction domains in each molecule that are highly conserved among most known subunits. However, the interaction domain within CyCaalpha1, an alpha1 subunit cloned from the jellyfish Cyanea capillata, matches the canonical sequence of the alpha1 interaction domain at only four of nine sites. We have now cloned a cDNA from Cyanea neuromuscular tissue that encodes a Ca2+ channel beta subunit. The subunit, named CyCabeta, shares 47-54% identity with vertebrate beta subunit isoforms, but is most highly conserved within its interaction domain. Coexpression of CyCabeta with CyCaalpha1 in Xenopus oocytes increases the amplitude of the CyCaalpha1 current and shifts its activation to more hyperpolarized potentials. These responses are mimicked by coexpression of the rat beta2a subunit, demonstrating that the alpha1 beta interaction is functionally conserved between cnidarians and mammals. CyCabeta also markedly accelerates the rate of recovery of CyCaalpha1 from inactivation, an action that is modestly duplicated by beta2a and may represent an additional mechanism by which beta subunit isoforms differentially modulate alpha1 subunits. These findings establish that limited conservation within the alpha1 interaction domain is sufficient to allow full modulation by a beta subunit, as well as altered regulation by different beta isoforms.
Asunto(s)
Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Escifozoos/genética , Escifozoos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio Tipo L/química , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/genética , Femenino , Expresión Génica , Técnicas In Vitro , Datos de Secuencia Molecular , Oocitos/metabolismo , Estructura Cuaternaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.
Asunto(s)
Canales de Calcio/genética , Activación del Canal Iónico , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio/efectos de los fármacos , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Escifozoos , Homología de Secuencia de AminoácidoRESUMEN
The neuromuscular sodium currents of early invertebrates such as platyhelminths display distinctive kinetic and pharmacological properties. We have cloned a cDNA from the horseshoe crab flatworm Bdelloura candida that encodes a protein homologous to the primary subunit of voltage-gated sodium channels. The B. candida protein, named BdNa1, exhibits amino acid identity of 40-47% to sodium channels of vertebrates and higher invertebrates. BdNa1 has the multidomain structure characteristic of sodium channels, and is most highly conserved in the hydrophobic transmembrane segments and the regions that form the pore of the channel. Northern blot analysis confirms the presence of a 5.4 kb BdNa1 transcript in B. candida tissue. The information provided by analysis of the BdNa1 sequence offers insight into the physiology of platyhelminth sodium currents.