Calmodulin regulation of voltage-gated calcium and sodium channels.

摘要

Voltage-gated ion channels are integral membrane proteins that control the flux of ions across the cell membrane, generating electrical signals. Over the past decade, calmodulin (CaM), a ubiquitous Ca2+-binding protein has emerged as a novel ion channel subunit that modulates channel function in a Ca2+-dependent manner. In this thesis, I investigated the role of CaM in the regulation of voltage-gated Ca2+ and Na+ channels.; Inactivation of the voltage-gated L-type Ca2+ channel (Cav1.2) is accelerated by CaM in process known as Ca2+ -dependent inactivation (CDI). In this study, I reconstituted the Ca2+ sensor in vitro, which consists of the CaM binding pocket of alpha1C, the pore forming subunit of the channel, and CaM. Using this complex, I identified the molecular determinants of CaM binding as well as the Ca2+-dependent conformational change that underlies inactivation. CDI was abolished by CaM mutants that blocked the conformational change and CDI was reduced by a mutation in the channel that prevented the foil conformational change. In addition, Ile1654 in the CaM-binding IQ motif of alpha1C forms the link between the Ca2+-sensor and the downstream inactivation machinery, using the alpha1C EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha1C I-II intracellular linker.; Identification of a CaM binding domain in the voltage-gated Ca 2+ channel has led to the investigation of whether other voltage-gated channels are regulated likewise. Ca2+ has been proposed to regulate voltage-gated Na+ channels (Nav1.x) through the action of CaM bound to a consensus CaM binding IQ motif or through direct binding to a paired EF hand motif in the Nav1.5 C-terminus. In contrast to a previous report, I found that Ca2+ does not bind directly to the Nav1.5 C-terminus. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C-terminus in a manner that differs significantly from CaM regulation of Cav1.2. This type of interaction between CaM and a sodium channel C-terminus is not unique to Nav1.5. In Nav1.2 or Nav1.5, CaM binds to a localized region containing the IQ motif, and the in vitro complex of CaM and the Nav1 C-termini does not undergo the large Ca2+-dependent conformational change seen in the CaM/Cav1.2 complex. Further, CaM binding to Nav1 C-termini lowers Ca2+ binding affinity and cooperativity among the CaM binding sites compared to CaM alone. Mutations in the localized CaM binding region altered the properties of the CaM/Nav1 complex. In particular, a mutation in Nav1.2 linked to familial autism (R1902C) allowed the complex to undergo a moderate conformational change. Identification of CaM's role in the regulation of Nav1 function has been elusive; however, in Nav1.5, I found that CaM modulates the C-terminal interaction with the inactivating particle III-IV linker that has been suggested to be involved in the stabilization of the inactivation gate and prevention of cardiac arrhythmias.; Together, these studies demonstrate that CaM modulates Cav1 and Nav1 function through unique molecular mechanisms.