Structural Basis for Calcium Modulation of the Cardiac Sodium Channel

Abstract

Voltage gated sodium channels drive the upstroke of the cardiac action potential and then undergo a fast inactivation process which is essential for the termination of their contribution to excitability and for the maintenance of cardiac rhythms. The degree to which the channel opens and inactivates relies on many cellular factors. Increased cytoplasmic calcium, for instance, results in a rightward shifted steady-state inactivation relationship for the cardiac Nav1.5 isoform, resulting in more channels being available to contribute to the action potential. We have previously shown using isothermal titration calorimetry (ITC) that Ca2+ /calmodulin can bind the sodium channel inactivation gate comprised of the cytoplasmic linker between domains III and IV in a calcium dependent manner (Kd at 1mM free [Ca2+]=3uM). To further examine this interaction in greater detail, we have determined a high-resolution (1.35A) crystal structure of the rigid portion of the two proteins in complex. This new structure confirms our previous findings that tyrosine 1494 acts as a major interaction point with the calmodulin C-lobe. With this structural data in hand we have designed channel mutations that increase (Kd=600nM) or decrease (approximately Kd=30uM) the affinity of calmodulin to the inactivation gate. We then analyzed the effect of these novel mutations on channel gating via patch clamp electrophysiology to further investigate the mechanisms of calcium regulation of the cardiac sodium channel. We account for our observations obtained from ITC and patch clamp experiments with a kinetic model describing calcium modulation of cardiac sodium channels. Our results suggest that the inactivation gate is a key molecular determinant in the regulation of cardiac sodium channels by calcium.