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_v1.3/Na_v1.1 (ICA-121431, IC₅₀, 19 nM) or Na_v1.7 (PF-04856264, IC₅₀, 28 nM) vs. other TTX-sensitive or resistant (i.e., Na_v1.5) 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_v1.3 and Na_v1.7 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_v1.3)/D1586 (Na_v1.7)] that is important for Site 3 α-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.Voltage-gated sodium (Na_v) channels play an important role in the generation and propagation of electrical signals in excitable cells (1–3). Eukaryotic Na_v channels are heteromeric membrane proteins composed of a pore-forming α-subunit and auxiliary β-subunits (3, 4). The mammalian genome encodes nine distinct α (Na_v1.1–1.9) and four β subunits (3). The α-subunit comprises four homologous domains (D1–D4), each of which contains six transmembrane segments (S1–S6) (3–5). The S5 and S6 segments form the central pore separated by the SS1 and SS2 segments, which form the ion selectivity filter at its extracellular end. The S1 to S4 segments form the voltage sensor (3–5).Both naturally occurring and synthetic pharmacological modulators of sodium channel have been identified (6–11), and for many, their site of interaction has been defined. For example, the marine toxin TTX inhibits several Na_v subtypes by interacting with amino acid residues within the SS1–SS2 segments that define the outer pore of the channel (12, 13). In contrast, the polypeptide α- and β-scorpion venom toxins, which enhance sodium channel activation or delay inactivation, and spider venom toxin sodium channel inhibitors like Protx II interact with specific residues on the S1–S4 voltage sensor regions within homologous Domain 2 (i.e., β-scorpion toxins, Protx II) or Domain 4 (i.e., α-scorpion toxins, anemone toxins) of the channel (8, 14–18). Many synthetic small molecule inhibitors of Na_v channels, including local anesthetic, antiepileptic, and antiarrhythmic agents, are believed to interact with amino acid residues within the S6 segment in Domain 4, which forms part of the pore lining and is structurally highly conserved across subclasses of mammalian Na_v channels (11, 19–22). This structural homology probably accounts for many clinically used local anesthetics and related antiepileptic and antiarrhythmic inhibitors, exhibiting little or no selectivity across the nine subtypes of mammalian Na_v channels (23). In the clinic this absence of subtype selectivity can result in toxicities associated with unwanted interactions with off-target Na_v channels (e.g., cardiac toxicity due to inhibition of cardiac Na_v1.5 channels) (24, 25). Therefore, because of their importance in normal physiology and pathophysiology, identification of selective pharmacological modulators of Na_v channels is of considerable interest to the scientific and medical communities (9, 23, 26–29). For example, in addition to the therapeutic utility of sodium channel inhibitors described above, there has been recent interest in potentially targeting inhibition of specific Na_v channel subtypes (i.e., Na_v1.7, Na_v1.8, and Na_v1.3) for the treatment of pain (9, 30–32).The present study describes the characterization of a class of subtype selective sodium channel inhibitor that interacts with a unique site on the voltage sensor region of homologous Domain 4. This inhibitory interaction site differs from previously reported inhibitor binding sites for TTX and local anesthetic-like modulators (11, 12).