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 ((mu)1/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, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM 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 approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.
Wang
S Y
SY
Department of Biology, State University of New York at Albany, Albany, New York, USA.
Barile
M
M
Wang
G K
GK
D019943:Amino Acid Substitution; D000779:Anesthetics, Local / Q000494:pharmacology*; D001496:Batrachotoxins / Q000494:pharmacology*; D002045:Bupivacaine / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010641:Phenotype; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D000779:Anesthetics, Local; D001496:Batrachotoxins; D015222:Sodium Channels; D002045:Bupivacaine