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Interactions of the C-11 Hydroxyl of Tetrodotoxin with the Sodium Channel Outer Vestibule

2003; Elsevier BV; Volume: 84; Issue: 1 Linguagem: Inglês

10.1016/s0006-3495(03)74849-8

1542-0086

Gaurav Choudhary, Mari Yotsu‐Yamashita, Lisa Shang, Takeshi Yasumoto, Samuel C. Dudley,

Nicotinic Acetylcholine Receptors Study

The highly selective sodium channel blocker, tetrodotoxin (TTX) has been instrumental in characterization of voltage-gated sodium channels. TTX occludes the ion-permeation pathway at the outer vestibule of the channel. In addition to a critical guanidinium group, TTX possesses six hydroxyl groups, which appear to be important for toxin block. The nature of their interactions with the outer vestibule remains debatable, however. The C-11 hydroxyl (C-11 OH) has been proposed to interact with the channel through a hydrogen bond to a carboxyl group, possibly from domain IV. On the other hand, previous experiments suggest that TTX interacts most strongly with pore loops of domains I and II. Energetic localization of the C-11 OH was undertaken by thermodynamic mutant cycle analysis assessing the dependence of the effects of mutations of the adult rat skeletal muscle Na(+) channel (rNa(v)1.4) and the presence of C-11 OH on toxin IC(50). Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents were measured by two-electrode voltage clamp. Toxin blocking efficacy was determined by recording the reduction in current upon toxin exposure. Mutant cycle analysis revealed that the maximum interaction of the C-11 OH was with domain IV residue D1532 (DeltaDeltaG: 1.0 kcal/mol). Furthermore, C-11 OH had significantly less interaction with several domain I, II, and III residues. The pattern of interactions suggested that C-11 was closest to domain IV, probably involved in a hydrogen bond with the domain IV carboxyl group. Incorporating this data, a new molecular model of TTX binding is proposed.