Calcium modification of voltage gated sodium channels

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PROJECT SUMMARY Voltage-gated ion channels are essential for action potentials in excitable cells located throughout the body (central nervous system, smooth muscle, heart and skeletal muscle). Loss of, improper, or untimely function, can each cause or contribute to disease. Many individual point mutations in the genes of ion channel or accessory proteins have been associated with disease, some of which can be life threatening. Many disease- associated mutations are at or near accessory protein binding sites. Therefore, significant effort has been put forth by many investigators to characterize mechanisms of ion channel gating modification. It is well established that Ca2+ can alter ion channel function, and the Ca2+ sensing protein calmodulin (CaM) has a prominent role in these processes. Structural investigations have identified many distinct CaM-ion channel interactions; however, the posited physiological function and interpretation of this data is often controversial. Early studies relied on measuring ion channel function in the absence or presence of Ca2+ and this has generated seemingly disparate results. Subsequent investigation revealed the mechanism(s) of Ca2+- driven modification are complex and can involve multiple accessory proteins. I previously identified a high-affinity interaction between CaM and part of a voltage-gated sodium channel that is directly responsible for inactivating conduction. I leveraged my in-depth structural characterization to impair the CaM interaction without conferring additional modification to channel function. This is a notable accomplishment given this part of the channel undergoes rapid conformational change during each functional cycle. Because of this, I could for the first time clearly attribute modified sodium channel function to reduced CaM binding. My data demonstrate that sodium channels with this reduced CaM interaction require longer to recover from the inactivated state. Considering my structure/function findings with literature suggests a paradigm of CaM Facilitated Recovery from Inactivation (CFRI). As demonstrated in my papers and scientific data, CaM engages the inactivation gate of several sodium channel isoforms with high affinity, suggesting a unique model of regulation. My findings are in direct conflict with other reports that posit models of CaM Dependent Inactivation (CDI) and [Ca2+] insensitivity. These opposing models arise from knowledge gaps regarding (i) the kinetic rates of CaM interactions and (ii) the precise role of each CaM interaction in an excitable cell that contains oscillating [Ca2+]. My proposal addresses these knowledge gaps by uniquely combining structural biology, stopped-flow kinetics, and electrophysiology to dissect the roles of the CaM-ion channel interactions in excitable cells. Importantly, we then leverage this knowledge to design custom small molecules (SAR by NMR approach) that alter the kinetics of accessory protein interactions, with a goal of tuning channel gating. This work will test models of Ca2+ modification of ion channel function, and explore novel strategies for treating channelopathies.