Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin

Batrachotoxin (BTX) is a steroidal alkaloid that causes Naf channels to open persistently, This toxin has been used widely as a tool for studying Na+ channel gating processes and for estimating Na+ channel density, In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine --> lysine substitution of the rat muscle Na+ channel alpha-subunit, mu 1-N434K, renders the channel completely insensitive to 5 mu M BTX when expressed in mammalian cells, This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, mu 1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified mu 1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional, This finding implies that the mu 1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the mu 1-434 position apparently diminishes the efficacy of BTX on eliminating Na+ channel inactivation, Mutants of two adjacent residues, mu 1-I433K and mu 1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the mu 1-I433, mu 1-N434, and mu 1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.