Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule

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

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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 (rNav1.4) and the presence of C-11 OH on toxin IC50. 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 (ΔΔG: 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.

Original languageEnglish
Pages (from-to)287-294
Number of pages8
JournalBiophysical Journal
Issue number1
Publication statusPublished - 2003 Jan 1

ASJC Scopus subject areas

  • Biophysics


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