The voltage-gated sodium channel Na v

Inherited mutations in voltage-gated sodium channels (VGSCs; or Nav) cause many disorders of excitability, including epilepsy, chronic pain, myotonia, and cardiac arrhythmias. Understanding the functional consequences of the disease-causing mutations is likely to provide invaluable insight into the roles that VGSCs play in normal and abnormal excitability. Here, we sought to test the hypothesis that disease-causing mutations lead to increased resurgent currents, unusual sodium currents that have not previously been implicated in disorders of excitability. We demonstrated that a paroxysmal extreme pain disorder (PEPD) mutation in the human peripheral neuronal sodium channel Nav1.7, a paramyotonia congenita (PMC) mutation in the human skeletal muscle sodium channel Nav1.4, and a long-QT3/SIDS mutation in the human cardiac sodium channel Nav1.5 all substantially increased the amplitude of resurgent sodium currents in an optimized adult rat-derived dorsal root ganglion neuronal expression system. Computer simulations indicated that resurgent currents associated with the Nav1.7 mutation could induce high-frequency action potential firing in nociceptive neurons and that resurgent currents associated with the Nav1.5 mutation could broaden the action potential in cardiac myocytes. These effects are consistent with the pathophysiology associated with the respective channelopathies. Our results indicate that resurgent currents are associated with multiple channelopathies and are likely to be important contributors to neuronal and muscle disorders of excitability.

Mutations in the TTX-sensitive voltage-gated sodium channel subtype Na v 1.7 have been implicated in the painful inherited neuropathy, hereditary erythromelalgia. Hereditary erythromelalgia can be difficult to treat and, although sodium channels are targeted by local anaesthetics such as lidocaine (lignocaine), some patients do not respond to treatment with local anaesthetics. This study examined electrophysiological differences in Na v 1.7 caused by a hereditary erythromelalgia mutation (N395K) that lies within the local anaesthetic binding site of the channel. The N395K mutation produced a hyperpolarized voltage dependence of activation, slower kinetics of deactivation, and impaired steady-state slow inactivation. Computer simulations indicate that the shift in activation is the major determinant of the hyperexcitability induced by erythromelalgia mutations in sensory neurons, but that changes in slow inactivation can modulate the overall impact on excitability. This study also investigated lidocaine inhibition of the Na v 1.7-N395K channel. We show that the N395K mutation attenuates the inhibitory effects of lidocaine on both resting and inactivated Na v 1.7. The IC 50 for lidocaine was estimated at 500 μM for inactivated wild-type Na v 1.7 and 2.8 mM for inactivated Na v 1.7-N395K. The N395K mutation also significantly reduced use-dependent inhibition of lidocaine on Na v 1.7 current. In contrast, a different hereditary erythromelalgia mutation (F216S), not located in the local anaesthetic binding site, had no effect on lidocaine inhibition of Na v 1.7 current. Our observation of reduced lidocaine inhibition on Na v 1.7-N395K shows that the residue N395 is critical for lidocaine binding to Na v 1.7 and suggests that the response of individuals with hereditary erythromelalgia to lidocaine treatment may be determined, at least in part, by their specific genotype.

Single-point missense mutations in the peripheral neuronal voltage-gated sodium channel Nav1.7 are implicated in the painful inherited neuropathy paroxysmal extreme pain disorder (PEPD). The Nav1.7 PEPD mutations are located in regions of the channel suggested to play important roles in fast inactivation. PEPD mutations in the putative inactivation gate have been reported to significantly impair fast inactivation, resulting in pronounced persistent currents. However, PEPD mutations in the S4-S5 linker of domain 3 (D3/S4-S5) had not been characterized and the roles of specific residues in this linker in channel gating are unclear. We functionally characterized two of the D3/S4-S5 PEPD mutations (V1298F and V1299F) and compared their effects on gating to an adjacent non-PEPD mutation (V1300F) and the I1461T PEPD mutation, located in the putative inactivation gate. The primary effect of the V1298F and V1299F mutations is to shift the voltage dependence of fast inactivation by ∼20 mV in the depolarizing direction. We observed a similar effect with the PEPD mutation I1461T. Interestingly, while all three PEPD mutations increased persistent currents, the relative amplitudes (∼6% of peak) were much smaller than previously reported for the I1461T mutation. In contrast, the main effect of the V1300F mutation was a depolarizing shift in the voltage dependence of activation. These data demonstrate that (1) mutations within D3/S4-S5 affect inactivation of Nav1.7 in a residue-specific manner and (2) disruption of the fast-inactivated state by PEPD mutations can be more moderate than previously indicated, which has important implications for the pathophysiology of PEPD.

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxinbinding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.Voltage-gated sodium channels (VGSCs) 3 play critical roles in the generation and propagation of action potentials. Nine VGSC ␣ subunit subtypes (Nav1.1-1.9) have been cloned and characterized from mammals (1). These subtypes are expressed in different excitable tissues and are involved in distinct physiological functions such as neurotransmitter release, muscle contraction, secretion, and pain sensation (2-4). The nine ␣ subunits share a common four-domain (DI-DIV) structure, in which each domain has six transmembrane segments (S1-S6) (1). Based on distinct functional behaviors during channel activity, the six transmembrane segments are generally separated into two structural components (5). The central pore module is the basis for the ion conduction pathway and is formed by the S5-S6 region of the channel. The voltage sensor modules are formed by the S1-S4 regions and are essentially independent structures that directly respond to changes in the transmembrane potential with conformational alterations that are coupled to opening and closing of the pore module. VGSCs undergo voltage-dependent activation subsequently followed by fast inactivation through successive activities of the voltage sensors in the four domains. The S4 segments in the voltage sensors of DI, DII, and DIII are determinants of channel activation, whereas that of DIV is predominantly involved in channel inactivation (5-8). The four membrane-spanning segments (S1-S4) of the voltage sensors of mammal VGSCs exhibit high sequence similarity in the four domains, but the amino acids and sequenc...