RESUMEN
Voltage-gated Ca(2+) channel beta (Ca(v)beta) subunits have a highly conserved core consisting of interacting Src homology 3 and guanylate kinase domains, and are postulated to exert their effects through AID, the major interaction site in the pore-forming alpha(1) subunit. This stereotypical interaction does not explain how individual Ca(v)beta subunits modulate alpha(1) subunits differentially. Here we show that AID is neither necessary nor sufficient for critical Ca(v)beta regulatory properties. Complete modulation depends on additional contacts that are exclusive of AID and not revealed in recent crystal structures. These data offer a new context for understanding Ca(v)beta modulation, suggesting that the AID interaction orients the Ca(v)beta core so as to permit additional isoform-specific Ca(v)alpha(1)-Ca(v)beta interactions that underlie the particular regulation seen with each Ca(v)alpha(1)-Ca(v)beta pair, rather than as the main site of regulation.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Animales , Canales de Calcio/genética , Electrofisiología , Guanilato-Quinasas , Cinética , Nucleósido-Fosfato Quinasa/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Xenopus laevis , Dominios Homologos srcRESUMEN
Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.
Asunto(s)
Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio/fisiología , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Bovinos , Femenino , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína/genética , Ratas , XenopusRESUMEN
beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.
Asunto(s)
Canales de Calcio/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Subunidades de Proteína/fisiología , Animales , Canales de Calcio/química , Conductividad Eléctrica , Electrofisiología , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Guanilato-Quinasas , Humanos , Cinética , Potenciales de la Membrana , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Inhibición Neural , Nucleósido-Fosfato Quinasa/fisiología , Oocitos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Conejos , Ratas , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Xenopus , LevadurasRESUMEN
Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca(2+)-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca(2+)-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.