title: Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule.
abstract: 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.
authors.affiliation: Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Choudhary
authors.forename: Mari
authors.initials: M
authors.lastname: Yotsu-Yamashita
authors.forename: Lisa
authors.initials: L
authors.lastname: Shang
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yasumoto
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Dudley
mesh_terms: D000818:Animals; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D003001:Cloning, Molecular; D017665:Hydroxyl Radical / Q000737:chemistry; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D013779:Tetrodotoxin / Q000737:chemistry* / Q000494:pharmacology* / Q000502:physiology; D014981:Xenopus / Q000502:physiology
chemical_list: D011994:Recombinant Proteins; D015222:Sodium Channels; D017665:Hydroxyl Radical; D013779:Tetrodotoxin; D002244:Carbon