Calmodulin Mediates Ca2+ Sensitivity of Sodium Channels

Abstract

Ca2؉ has been proposed to regulate Na ؉ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Na v 1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca 2؉ confers sensitivity are poorly understood. Studies on the homologous Ca v 1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na ؉ channels. In contrast to previous reports, we found that Ca 2؉ does not bind directly to Na ؉ channel C termini. Rather, Ca 2؉ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Ca v 1.2. In Na v 1.2 or Na v 1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca -dependent conformational change in Na v 1.2 C terminus⅐CaM complex that was absent in the wild-type complex. In Na v 1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca 2؉

Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

“…2, B and C) is consistent with numerous studies showing that mutations in the two cytoplasmic domains have concordant effects on the voltage dependence of inactivation, and suggests functional interaction between these regions (9, 29 -31). Moreover, evidence has accumulated in support of interactions between CaM, the C terminus, and the DIII-DIV linker (13,24).…”

“…In addition, the DIII-DIV linker has recently been implicated as a site for CaM interaction (13,14,24). Moreover, it has been proposed that CaM is required to stabilize the interaction of the C terminus with the DIII-DIV linker (13).…”

“…in the C terminus (8,12,13). This domain exhibits multiple CaM binding modes, which are influenced by [Ca 2ϩ ] i (14), as well as an interaction with a C-terminal EF-hand domain that directly binds the IQ domain (15).…”

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

“…Biochemical Evidence for an H1/H4 Hydrophobic InterfaceThe predicted EF-hand pair in the Na V 1.5 COOH terminus contains a single tryptophan, Trp 1798 , which is important not only because our model predicts it to be integral to the hydrophobic interface but also because its intrinsic fluorescence can be used to probe and report the nature of the environment in which it is located (13,24,25). We thus prepared a GST fusion protein consisting of residues predicted to form the EF-hand pair (residues 1786 -1863) and carried out experiments using Trp 1798 as a reporter of its environment.…”

“…Inherited mutations of the III/IV linker in the cardiac Na ϩ channel can disrupt fast inactivation, resulting in sustained current (I SUS ), which can cause long QT syndrome variant 3 (5). However, the Na V 1.5 COOH terminus also has been shown to play a role in inactivation both through chimeric studies (7), through the characterization of several disease-linked mutations found in the C terminus (8 -11), and by direct biochemical evidence for COOH terminus interactions with the cytoplasmic peptide that links domains III and IV of the ␣ subunit (III-IV linker) (12,13). …”