Investigating Ca2+-Dependent Regulation of Sodium Channels via Thermodynamic and Structural Analysis of Nav1.4 and Nav1.5 Carboxy Tail Interactions with Calmodulin

Abstract

Voltage-gated sodium channels (Nav) are essential for cardiac and skeletal muscle function. Channelopathic mutations in cardiac sodium channel (Nav1.5) and skeletal muscle sodium channel (Nav1.4), particularly in their cytoplasmic carboxy tails (CTNav), give rise to numerous arrhythmias and myotonias. In Nav regulation, CTNav partners with the Ca2+-sensing protein calmodulin (CaM). We aim to understand how CaM regulates channel function. Ca2+-free CaM (apoCaM) is a regulatory modulator of Nav, and apoCaM bound to the CTNav increases the channel's open probability. CaM also participates in the Ca2+-dependent inactivation (CDI) of Nav1.4. In contrast, Nav1.5 does not show CDI and no role of Ca2+-CaM is known in Nav1.5. To understand the Ca2+-control of CaM regulation of these sodium channels we have collected binding data of CTNavs with CaM, in the presence and absence of Ca2+. Binding data of CTNavs with CaM mutants with Ca2+-binding knocked out in either of CaM's functional domains (lobes) have also been collected to understand the distinct roles of CaM's lobes. Collectively, these binding data have allowed us to predict CTNav and CaM populations as a function of Ca2+ concentration. To gain information on the structural changes induced by Ca2+ on the CTNav-CaM complexes, we conducted small angle scattering (SAXS) experiments on CTNav1.4-CaM and CTNav1.5-CaM in the presence and absence of Ca2+. The distance distribution function, P(r), shows changes in both complexes upon addition of Ca2+. Computational modeling of flexible forms of the complexes reveals conformations of CTNav-CaM, in the presence and absence of Ca2+, that are compatible with our SAXS data. The molecular envelope of the CTNav1.5-apoCaM matches well with the apoCaM-CTNav1.5 crystal structure previously determined by our lab.