Disparate role of Na+ channel D2-S6 residues in batrachotoxin and local anesthetic action

Batrachotoxin (BTX) stabilizes the voltage-gated Na+ channels in their open conformation, whereas local anesthetics (LAs) block Na+ conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha -subunit Na+ channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na+ channel (mu1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na+ currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na+ currents during repetitive pulses. Two of these four mutants, mu1-N784K and mu1-L788K, were completely resistant to 5 muM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na+ channels (K-I) of similar to 10 muM or less, which suggests that mu1-N784 and mu1-L788 residues are not in close proximity to the LA binding site.