Calcium Dysregulation of Voltage-Gated Sodium Channels Harboring LQT3 Mutations

Abstract

Intracellular calcium ions modulate sodium channel inactivation by producing a depolarizing shift the steady-state inactivation equilibrium. We have recently proposed a mechanism for this effect by which direct Ca2+/calmodulin(CaM) binding to the inactivation gate increases the transient availability of channels in the action potential by shifting the steady-state inactivation. Interestingly, a crystal structure of Ca2+/CaM bound to the inactivation gate of the sodium channel pinpoints the position of four mutations (M1498T, K1500Δ, L1501V, G1502S) shown previously to underlie long QT3 syndrome, along a critical binding interface. We explored the possibility that these mutations, in addition to effects on channel gating, may alter calcium regulation of Nav1.5. This possibility was first tested directly with Isothermal Titration Calorimetry (ITC) to determine the binding parameters of purified proteins and then by patch-clamp electrophysiology of expressed wild-type and mutant channels. Interestingly, ITC experiments demonstrated that the mutations impacted Ca2+/CaM binding by altering the affinity of Ca2+/CaM for the inactivation gate. Channels carrying inherited mutation showed robust expression in HEK-293 cells with either modest or severe effects on channel gating, as expected for LQT3 mutations. However, in terms of Ca2+ regulation, the LQT3 mutations significantly altered the calcium-induced shift in steady-state inactivation compared to wild-type channels. The data suggest that calcium dysregulation of the voltage-gated sodium channel may contribute to the pathogenesis of LQT3 syndrome.