Modulation of sodium channel gating

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Voltage-gated sodium channels (NaVs) are responsible for the initiation and propagation of action potentials in many excitable cells. NaVs undergo a conformational change in response to changes in the electrical field across cell membranes. NaVs consist of a pore-forming and voltage-sensing alpha subunit, and 1 or 2 'auxillary' beta subunits. The structure of the alpha-subunit of NaVs includes four homologous domains with voltage-sensing domains (VSDs) and pore-forming domains (PDs), the latter of which combine to form a gated, water-filled pathway through which sodium ions flow down their electrochemical gradient. The VSDs are made of 4 membrane-spanning alpha-helices (S1-S4). The S4s in the VSD include a high concentration of positively-charged amino acids (AAs), lysines or arginines, in every third position. The positive AAs form salt bridges with negatively-charged AAs in the S2 and S3 segments of the VSDs. Upon depolarization of the electrical field, the VSDs rearrange such that the S4s rotate and translocate in the extracellular direction, making and breaking salt bridges with the negative AAs in S2 and S3. The 'outward' movement of the S4s creates a small current caused caused by the movement of positive charges within the electrical field. In voltage-clamp recordings, this so-called 'gating current' is typically obscured by the much larger ionic current caused by sodium channels moving through the pore. With a sufficiently large population of channels and the ionic current blocked with tetrodotoxin, however, gating currents can be measured and their properties quantified, giving a direct measure of VSD activity under a variety of conditions. NaVs are subject to post-translational modification, including co-association with beta subunits, phosphorylation by protein kinase A, protein kinase C, tyrosine kinase, and calmodulin-dependent protein kinase II, and by calmodulin-mediated calcium binding. The effects of these post-translational modification processes on ionic currents have been well-studied. However, the effects of post-translational modification on gating currents has not yet been described. In the present application, we propose to test the hypotheses that (1) co-association with beta subunits, (2) phosphorylation, and (3) calmodulin-mediated calcium binding alters biophysical properties of NaV gating currents and VSD movements. We will test these hypotheses using NaVs expressed in Xenopus oocytes, recorded using the cut-open oocyte voltage clamp (COVC) and voltage clamp fluorimetry (VCF). COVC allows gating currents to be measured and their biophysical properties quantified. VCF allows direct measurement of individual voltage-sensor translocation. Combined, these measurements permit a detailed analysis of VSD rearrangement and will allow us to answer the question: how is gating in NaVs modulated by post-translational modification?