Stereoselective inhibition of neuronal sodium channels by local anesthetics. Evidence for two sites of action?

The objective of this study was to determine if the "tonic," resting inhibition of Na+ channels by local anesthetics results from binding at a site different from that for "phasic," use-dependent inhibition. Stereoselective actions of four local anesthetics were examined in isolated frog peripheral nerve and single Na+ channels. Using the sucrose-gap method on desheathed nerves, four actions of local anesthetics were assayed: 1) tonic depression of compound action potentials at low stimulation frequency (one per minute); 2) phasic depression of the compound action potential during trains of stimulation at 5, 10, and 20 Hz; 3) competitive antagonism of the reversible Na+ channel activator veratridine assayed through the depolarization of the compound resting membrane potential; and 4) depression of the depolarization of the compound resting membrane potential initially induced by the irreversible channel activator batrachotoxin. For assays 1, 2, and 3, all local anesthetics showed a stereoselectivity, where rectus, or (+), enantiomers were more potent than sinister, or (-), enantiomers. In contrast, for the noncompetitive antagonism of veratridine's action and the depression of batrachotoxin-induced depolarization, also a noncompetitive interaction between anesthetic and activator, the (-) enantiomer was more potent than the corresponding (+) enantiomer. Blockade of single Na+ channels activated by batrachotoxin in planar lipid bilayers was also stereoselective for the (-) enantiomer. These findings, along with previously reported voltage-clamp results, can be applied to infer properties of a local anesthetic binding site in activator-free channels. Local anesthetic molecules with more sharply angled shapes have stronger stereoselectivities than less angled, more planar drugs. The inversion of the stereopotency induced by the activators can be explained by either of two mechanisms. There may be two binding sites for local anesthetics, one of high and one of low affinity and of opposite stereoselectivity; activators may change the conformation at the high affinity site, reducing its local anesthetic affinity below that of the usual low affinity site and thereby revealing the pharmacology of the weaker site. Alternatively, only a single binding site may exist and be conformationally altered by activators such that both anesthetic affinity and stereopotency are modified. In activator-free channels, however, a single, high-affinity binding site with a constant stereoselectivity can account for both tonic and phasic inhibition by local anesthetics.