INTERACTION OF BATRACHOTOXIN WITH THE LOCAL-ANESTHETIC RECEPTOR-SITE IN TRANSMEMBRANE SEGMENT IVS6 OF THE VOLTAGE-GATED SODIUM-CHANNEL

The voltage-gated sodium channel is the site of action of more than si x classes of neurotoxins and drugs that alter its function by interact ion with distinct, allosterically coupled receptor sites. Batrachotoxi n (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited alloster ically by local anesthetics, We have investigated the interaction of B TX with amino acid residues I1760, F1764, and Y1771, which form part o f local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1761 (mutant F1764A) r educes tritiated BTX-A-20-alpha-benzoate binding affinity, causing a 6 0-fold increase in K-d. Alanine substitution for I1760, which is adjac ent to F1764 in the predicted 1VS6 transmembrane alpha helix, causes o nly a 4-fold increase in Kd In contrast, mutant Y1771A shows no change in BTX binding affinity. For wildtype and mutant Y1771A, BTX shifted the voltage for half-maximal activation approximate to 40 mV in the hy perpolarizing direction and increased the percentage of noninactivatin g sodium current to approximate to 60%. In contrast, these BTX effects mere eliminated completely for the F1764A mutant and were reduced sub stantially for mutant I1760A. Our data suggest that the BTX receptor s ite shares overlapping but nonidentical molecular determinants with th e local anesthetic receptor site in transmembrane segment 1VS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain -interface allosteric model of ligand binding and action, as previousl y proposed for calcium agonist and antagonist drugs acting on L-type c alcium channels.