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.
Bibliographical noteFunding Information:
Dr. Samuel C. Dudley, Jr. is supported by a Scientist Development Award from the American Heart Association, Grant-In-Aid from the Southeast Affiliate of the American Heart Association, a Proctor and Gamble University Research Exploratory Award, and the National Institutes of Health (HL64828). Dr. Mari Yotsu-Yamashita is supported by Grants-In-Aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 13024210).