Access and Binding of Local Anesthetics in the Closed Sodium Channel

Abstract

Local anesthetics (LAs) are known to bind Na ϩ channels in the closed, open, and inactivated states and reach their binding sites via extracellular and intracellular access pathways. Despite intensive studies, no atomic-scale theory is available to explain the diverse experimental data on the LA actions. Here we attempt to contribute to this theory by simulating access and binding of LAs in the KcsA-based homology model of the closed Na ϩ channel. We used Monte Carlo minimizations to model the channel with representative local anesthetics N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium (QX-314), cocaine, and tetracaine. We found the nucleophilic central cavity to be a common binding region for the ammonium group of LAs, whose aromatic group can extend either along the pore axis (vertical binding mode) or to the III/IV domain interface (horizontal binding mode). The vertical mode was earlier predicted for the open channel, but only the horizontal mode is consistent with mutational data on the closed-channel block.

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

“…The multi-MCM protocol (9) was used to dock TTX and impose specific distance constraints. No specific energy terms were used for cation-interactions, which were accounted for due to partial negative charges at the aromatic carbons (15). Further details of methodology can be found elsewhere (9).…”

“…Mutation I 4p46 V creates a void of a methyl group size in the tight intrarepeat contact between the P-helix and the ascending limb. P-helices in our models are practically immobile due to several strong contacts with S6 and S5 helices (15). To fill the void, glycine G 4p51 would move toward valine V 4p46 and pull the ascending limb of repeat IV away from the pore axis.…”

“…Alanine substitutions of W 4p52 alter the ionic selectivity of sodium channels (19). Cysteine substitution of W 4p52 abolishes the NaV1.4 block by lidocaine (20), a local anesthetic that binds in the inner pore (15). W 4p52 was suggested to directly interact with the local anesthetic bound in the inner pore (20), but in view of our models of sodium channels, direct contact of W 4p52 with the local anesthetic molecule is unlikely, suggesting that the mutation W 4p52 C affects the drug binding allosterically.…”

Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

“…The ZMM molecular modeling program has been used in all calculations. The BgNa v 1-1a, Kv1.2, and KcsA sequences were aligned (Table 1) as before (3,37). The extracellular loops, which are far from BTX-sensing residues, were not included in the model.…”

“…The extracellular linkers connecting S5 and S6 helices form the four re-entrant P-loops, which contain the selectivity filter residues. In the absence of x-ray structures of Na V channels, their homology models, which are based on x-ray structures of potassium channels, are used to explain structure-activity relationships of various sodium channel ligands, including local anesthetics (1)(2)(3)(4), steroidal activators (5)(6)(7)(8), and pyrethroid insecticides (9,10). Recent reinterpretation of data on substituted cysteine accessibility of the Ca V 2.1 channel (11) in view of a the channel homology model, which is based on the x-ray structure of the open voltage-gated potassium channel Kv1.2 (12), further supports a general similarity of the inner pore architecture in different voltage-gated cationic channels (13).…”