Calcium modification of voltage gated sodium channels

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PROJECT SUMMARY Voltage-gated sodium channels (NaVs) 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 NaV or accessory proteins have been associated with disease; some of which can be life threatening. Many disease associated mutations are located at or are near NaV accessory protein binding sites; therefore significant effort has been put forth by many investigators to characterize the mechanisms that underlie ion channel gating modification and in physiology and disease. It is well established that Ca2+ alters NaV function, and the Ca2+ sensing protein Calmodulin (CaM) has a prominent role in this process. Structural investigations have identified several distinct CaM-NaV interactions. However, the posited physiological function and interpretation of data are controversial. Early studies relied on measuring NaV function in the absence or presence of Ca2+ and generated seemingly disparate results. Subsequent investigation revealed the mechanism(s) of Ca2+driven NaV modification are complex and involve multiple accessory proteins, thereby rendering much of the data ambiguous. Recently, I identified a high-affinity interaction between CaM and part of NaV that is directly responsible for inactivating NaV conduction. I was able to utilize my in-depth structural characterization to impair the interaction without conferring additional modification to NaV 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 NaV function to reduced CaM binding. My data demonstrate that channels with this reduced CaM interaction require longer to recover from the inactivated state. Considering my structure / function findings with available literature suggest a paradigm of CaM Facilitated Recovery from Inactivation (CFRI). As demonstrated in my recent papers and preliminary data, CaM engages several NaV isoforms with high affinity, suggesting a universal 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-NaV interactions in excitable cells. I will then explore if I can alter the kinetics of specific interactions by engineering a small molecule probe. This work will test CFRI (physiology and disease), as well as explore novel strategies for treating NaV channelopathies.