Abstract

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 α-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 (μ1/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, μ1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na⁺ currents during repetitive pulses. Two of these four mutants, μ1-N784K and μ1-L788K, were completely resistant to 5 μM 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 ∼10 μM or less, which suggests that μ1-N784 and μ1-L788 residues are not in close proximity to the LA binding site.