Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels

Voltage-gated sodium (Na-v) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Na-v channel inhibitors that exhibit up to 1,000-fold selectivity for human Na(v)1.3/Na(v)1.1 (ICA-121431, IC50, 19 nM) or Na(v)1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Na(v)1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the extracellular facing regions of the S2 and S3 transmembrane segments of Na(v)1.3 and Na(v)1.7 seem to be major determinants of Na-v subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Na(v)1.3)/D1586 (Na(v)1.7)] that is important for Site 3 alpha-scorpion and anemone polypeptide toxin modulators of Na-v channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.