RESUMEN
L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo P/química , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/química , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/metabolismo , Canales de Calcio Tipo R/química , Canales de Calcio Tipo R/genética , Canales de Calcio Tipo R/metabolismo , Bovinos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Alternative splicing of the P/Q-type channel (Ca(V)2.1) promises customization of the computational repertoire of neurons. Here we report that concerted splicing of its main alpha1A subunit, at both an EF-hand-like domain and the channel C terminus, controls the form of Ca2+-dependent facilitation (CDF), an activity-dependent enhancement of channel opening that is triggered by calmodulin. In recombinant channels, such alternative splicing switches CDF among three modes: (1) completely "ON" and driven by local Ca2+ influx through individual channels, (2) completely "OFF," and (3) partially OFF but inducible by elevated global Ca2+ influx. Conversion from modes 1 to 3 represents an unprecedented dimension of control. The physiological function of these variants is likely important, because we find that the distribution of EF-hand splice variants is strikingly heterogeneous in the human brain, varying both across regions and during development.
Asunto(s)
Empalme Alternativo , Canales de Calcio/genética , Calmodulina/fisiología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo N , Canales de Calcio Tipo P , Canales de Calcio Tipo Q , Calmodulina/química , Calmodulina/farmacología , Línea Celular , Exones/genética , Transporte Iónico/efectos de los fármacos , Riñón , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , TransfecciónRESUMEN
P/Q-type (Ca(v)2.1) calcium channels support a host of Ca2+-driven neuronal functions in the mammalian brain. Alternative splicing of the main alpha1A (alpha1(2.1)) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human alpha1(2.1) gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca2+-dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.
Asunto(s)
Empalme Alternativo/genética , Canales de Calcio Tipo N/genética , Secuencia de Aminoácidos , Encéfalo/metabolismo , Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , Calmodulina/metabolismo , Línea Celular , Clonación Molecular , Bases de Datos de Ácidos Nucleicos , Exones , Humanos , Intrones , Riñón/citología , Riñón/metabolismo , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa/métodos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Análisis de Secuencia de ADN , TransfecciónRESUMEN
Engineered calmodulins (CaMs), rendered Ca2+-insensitive by mutations, function as dominant negatives in heterologous systems, and have revealed mechanisms of ion channel modulation by Ca2+/CaM. The use of these CaMs in native mammalian cells now emerges as a strategy to unmask the biology of such Ca2+ feedback. Here, we developed recombinant adenoviruses bearing engineered CaMs to facilitate their expression in adult heart cells, where Ca2+ regulation may be essential for moment-to-moment control of the heartbeat. Engineered CaMs not only eliminated the Ca2+-dependent inactivation of native calcium channels, but exposed an unexpectedly large impact of removing such feedback: the unprecedented (4- to 5-fold) prolongation of action potentials. This striking result recasts the basic paradigm for action-potential control and illustrates the promise of virally delivered engineered CaM to investigate the biology of numerous other CaM-signaling pathways.