Abstract 942: Calcium Sensing Domains In The Nav1.5 C-terminus Influence Channel Availability In Response To Changing Ca2+ Levels.
Sodium (Na) channels are dynamic molecules that change their conformational state (“gating”) in response to membrane potential and other stimuli. We have previously identified two mechanisms whereby intracellular Ca2+ is capable of directly modulating Na channel (NaV1.5) inactivation gating: A C-terminal IQ motif, which exhibits multiple Calmodulin (CaM) binding modes altered by calcium, and an immediately adjacent EF hand that directly binds calcium. Intracellular free Ca2+ rises in response to pathophysiologic stress. In this study we tested how changing Ca2+ levels influence the inactivation properties of channel variants carrying mutations in the C-terminal IQ motif and the EF hand. Voltage-dependent availability of heterologously expressed Nav1.5 was assessed by measuring the peak Na current at −20mV following 50 msec prepulses to potentials ranging from −130 to −10 mV. As we showed previously, WT Nav1.5 steady state availability is hyperpolarized when intracellular Ca2+ is depleted by adding BAPTA (V1/2= −79.5 ± 1.3 mV), compared to basal (1 μm) [Ca2+ ] (V1/2 = −69.6 ± 1.0 mV, P<0.001). A mutation linked to the Brugada Syndrome (A1924T), previously shown to diminish CaM binding, causes a hyperpolarizing shift in the availability curve (V1/2 = −75.1 ± 1.8 mV) compared to WT (V1/2 = −69.6 ± 1.0 mV, P<0.05) in basal Ca2+ but is yet further left-shifted when Ca2+ is depleted (V1/2 = −87.11 ± 1.1mV). Mutating selected residues to impair the Ca2+ binding EF hand motif (4X), or a region of the IQ motif that governs EF-hand-IQ interactions (IQ/AA), evokes a left-shift of the availability curve in basal Ca2+ (V1/2 = −85.79±2.19 mV and −82.41±0.79 mV, respectively) that recapitulates the response seen when Ca2+ is removed from the WT channel. Thus, either the reduced coupling of CaM to the C-terminal Ca2+ sensing IQ motif, or decreased Ca2+ affinity of the EF hand, result in a left-shift of channel inactivation. The findings suggest that CaM binding, and associated EF hand-IQ domain coupling within the C-terminus, work in concert to render the channel resistant to inactivation (increasing excitability) at typical resting membrane potentials and that changes in intracellular calcium are an essential mechanistic link between cellular stress and Na channel inactivation.