RESUMEN
A small expansion of a CAG repeat domain in exon 47 of the human CACNA1A gene, which codes for the pore-forming alpha1A subunit of P/Q-type Ca2+ channels, causes spinocerebellar ataxia type-6. Only the human alpha1A protein has been demonstrated to contain the poly(Q) tract, although this locus has also recently been detected in ape genomes. To our knowledge, no further information has been published on other mammal species. Here, we have cloned the full-length alpha1A subunit in a non-primate species, the cow. The results have made it possible to explore the exon organization of the bovine CACNA1A gene as well as the splice alpha1A isoforms expressed by bovine chromaffin cells. We found a splice variant of the protein that, as in humans, also contains a polymorphic poly(Q) tract. Based on this result and using data from different Genome Databases, we performed an interspecies comparison of exon 47 and discovered that the poly(Q) tract is present in all the species studied, with the exception of primitive fish and rodents. Our results provide insight into the evolution of the CAG repeat tract at the C-terminus coding region of the CACNA1A gene.
Asunto(s)
Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Ataxias Espinocerebelosas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio/genética , Bovinos , Células Cultivadas , Células Cromafines/metabolismo , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Evolución Molecular , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Repeticiones de TrinucleótidosRESUMEN
Although the specific interaction between synaptic protein SNAP-25 and the alpha1A subunit of the Cav2.1 channels, which conduct P/Q-type Ca2+ currents, has been confirmed in in vitro-translated proteins and brain membrane studies, the question of how native proteins can establish this association in situ in developing neurons remains to be elucidated. Here we report data regarding this interaction in bovine chromaffin cells natively expressing both proteins. The two carboxyl-terminal splice variants of the alpha1A subunit identified in these cells share a synaptic protein interaction ('synprint') site within the II/III loop segment and are immunodetected by a specific antibody against bovine alpha1A protein. Moreover, both alpha1A isoforms form part of the P/Q-channels-SNARE complexes in situ because they are coimmunoprecipitated from solubilized chromaffin cell membranes by a monoclonal SNAP-25 antibody. The distribution of alpha1A and SNAP-25 was studied in round or transdifferentiated chromaffin cells using confocal microscopy and specific antibodies: the two proteins are colocalized at the cell body membrane in both natural cell types. However, during the first stages of the cell transdifferentiation process, SNAP-25 migrates alone out to the developing growth cone and what will become the nerve endings and varicosities of the mature neurites; alpha1A follows and colocalizes to SNAP-25 in the now mature processes. These observations lead us to propose that the association between SNAP-25 and alpha1A during neuritogenesis might promote not only the efficient coupling of the exocytotic machinery but also the correct insertion of P/Q-type channels at specialized active zones in presynaptic neuronal terminals.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Células Cromafines/citología , Células Cromafines/fisiología , Neuritas/metabolismo , Subunidades de Proteína/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Animales , Secuencia de Bases , Northern Blotting/métodos , Western Blotting/métodos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/genética , Bovinos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Cromafines/clasificación , Células Cromafines/efectos de los fármacos , Dopamina beta-Hidroxilasa/metabolismo , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoprecipitación/métodos , Ratones , Microscopía Confocal/métodos , Modelos Moleculares , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Potasio/farmacología , Subunidades de Proteína/genética , ARN Mensajero/metabolismo , Conejos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia/métodos , Proteína 25 Asociada a Sinaptosomas/genéticaRESUMEN
Because the presence of a native plasmalemmal Na+/Ca2+ exchange (NCX) activity in Xenopus laevis oocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP3R) that controls intracellular Ca2+ in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP3Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca2+ mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+ -activated chloride current, IClCa, as a result of the Ca2+ rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent "Ca2+ extrusion" via NCX; and (v) blocking NCX enhanced the IClCa elicited by InsP3 but not by Ca2+ photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the "Ca2+ influx" but not for the serum-elicited "Ca2+ efflux" mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+ signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+ microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.
Asunto(s)
Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Oocitos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Tiourea/análogos & derivados , Secuencia de Aminoácidos , Animales , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Femenino , Expresión Génica , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , ARN Complementario/genética , Receptor Cross-Talk , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/genética , Tiourea/farmacología , Xenopus laevisRESUMEN
The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Gö6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Células Cromafines/enzimología , Proteínas de la Membrana/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Quinasas/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inhibidores de Proteínas Quinasas , Proteína 25 Asociada a SinaptosomasRESUMEN
Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.