TY - JOUR
T1 - Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule
AU - Choudhary, Gaurav
AU - Yotsu-Yamashita, Mari
AU - Shang, Lisa
AU - Yasumoto, Takeshi
AU - Dudley, Samuel C.
N1 - Funding 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).
PY - 2003/1/1
Y1 - 2003/1/1
N2 - 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.
AB - 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.
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U2 - 10.1016/S0006-3495(03)74849-8
DO - 10.1016/S0006-3495(03)74849-8
M3 - Article
C2 - 12524282
AN - SCOPUS:12244288289
VL - 84
SP - 287
EP - 294
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 1
ER -