RESUMO
Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Ativação do Canal Iônico/fisiologia , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Estimulação Elétrica , Ativação Enzimática , Feminino , Holoenzimas/fisiologia , Técnicas In Vitro , Modelos Biológicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Estrutura Terciária de Proteína , Subunidades Proteicas/fisiologia , Coelhos , Ratos , Xenopus laevisRESUMO
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.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Animais , Canais de Cálcio/genética , Eletrofisiologia , Guanilato Quinases , Cinética , Núcleosídeo-Fosfato Quinase/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Xenopus laevis , Domínios de Homologia de srcRESUMO
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.
