Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel

Abstract

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-␣-benzoate binding affinity, causing a 60-fold increase in K d . Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in K d . In contrast, mutant Y1771A shows no change in BTX binding affinity. For wildtype and mutant Y1771A, BTX shifted the voltage for halfmaximal activation Ϸ40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to Ϸ60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 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 previously proposed for calcium agonist and antagonist drugs acting on L-type calcium channels.Voltage-gated sodium channels are responsible for the rapid depolarization during the initial phase of the action potential in most excitable cells (1). The brain sodium channel consists of a pore-forming ␣ subunit of Ϸ260 kDa associated with a 36-kDa ␤1 subunit and a 33-kDa ␤2 subunit (reviewed in ref.2). The primary structure of the ␣ subunit is organized in four homologous domains (I-IV) that contain six transmembrane segments (S1-S6) and an additional membrane-associated segment between S5 and S6 and are connected by large intracellular loops (3, 4).Potent neurotoxins specifically bind to more than six distinct but allosterically coupled receptor sites on the voltage-gated sodium channel and modify its function with lethal consequences (2, 5-7). The sodium channel is also the site of action for local anesthetics and certain anticonvulsant and antiarrhythmic drugs that are used therapeutically to decrease sodium channel activity in syndromes of hyperexcitability, such as cardiac arrhythmia and epileptic seizure (1,8,9). Although these sodium channel binding sites are distinct, allosteric interactions among the sites modulate drug and toxin binding (10-15). Neurotoxin receptor site 2 binds neurotoxins that cause persistent activation of sodium channels, including batrachotoxin (BTX), veratridine, ac...

Single point mutations affect fatty acid block of human myocardial sodium channel α subunit Na + channels

“…The close functional association, as well as the close positional association, has further been demonstrated by the findings that point mutations in D4-S6, which inhibit the action of LAs on conductance of Na ϩ channels, also block the binding of BTX to the Na ϩ channel (21,22). Likewise, mutations in D1-S6, which block the binding of BTX, also block the inhibiting action of local anesthetics on the Na ϩ channel (23).…”

Synergistic interaction between two cockroach sodium channel mutations and a tobacco budworm sodium channel mutation in reducing channel sensitivity to a pyrethroid insecticide

“…The highaffinity-pyrethroid-binding site is located likely at the interface between domains I and II where the amino acid residues V409 and L993 reside, which is also suggested in the house fly sodium channel (Lee and Soderlund, 2001). Such domain interface models have been proposed for several classes of lipophilic neurotoxins acting on calcium and sodium channels (Hockerman et al, 1997;Linford et al, 1998). For example, sodium channel site 2 neurotoxin BTX activates sodium channels by binding to a site formed by unique amino acid determinants in IS6 and IVS6 (Linford et al, 1998).…”

“…Such domain interface models have been proposed for several classes of lipophilic neurotoxins acting on calcium and sodium channels (Hockerman et al, 1997;Linford et al, 1998). For example, sodium channel site 2 neurotoxin BTX activates sodium channels by binding to a site formed by unique amino acid determinants in IS6 and IVS6 (Linford et al, 1998).…”

Sodium Channel β4, a New Disulfide-Linked Auxiliary Subunit with Similarity to β2

“…Plasmids pCDM8-rIIA (containing the cDNA fragment of the full-length rat Na v 1.2a ␣ subunit; Linford et al, 1998) and pCDM8-rH1 (containing the cDNA of the full-length rat heart Na v 1.5 sodium channel ␣ subunit; Qu et al, 1994) have been described. The rSKM1 cDNA encoding the Na v 1.4 ␣ subunit (Featherstone et al, 1998) was a kind gift from Dr. Peter Ruben (Utah State University) and was subcloned into pCDM8 to produce pCDM8-rSKM1.…”