Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane

The widely used sodium channel blocker tetrodotoxin (TTX) is a compound tha t has six hydroxyl residues at the C-4, C-6, C-8, C-9, C-10, and C-11 posit ions in addition to a guanidinium group, which is positively charged in bio logical pH range. Thirteen analogs of this toxin with structural modificati ons involving one or more of these hydroxyls were examined on their affinit y to a rat brain membrane preparation, which is known to contain sodium cha nnels abundantly. The equilibrium dissociation constants associated with th e binding of mt and its analogs to the sodium channels were estimated, from their ability to inhibit the binding of [H-3]saxitoxin, as follows (in nM) : TTX, 1.8, chiriquitoxin, 1.0; 11-oxoTTX, 1.5; 11-norTTX-6,6-diol, 1.6; 11 -norTTX-6(S)-ol, 23; 11-norTTX-6(R)-ol, 31, 11-deoxyTTX, 37; 6-epiTTX, 39; 4-epiTTX, 68; 4,9-anhydroTTX, 180; TTX-8-O-hemisuccinate, >380; TTX-11-carb oxylic acid, >2300; tetrodonic acid, >3600; 5,6,11-trideoxyTTX, >5000. The reduction of the affinity observed with the analogs involving reduction or translocation of the hydroxyls at C-6 and C-11 is indicative of the contrib ution of these residues to the binding to sodium channels as hydrogen bond donors. The especially large value of the dissociation constant for TTX-11- carboxylic acid is consistent with the idea that the C-11-hydroxyl forms a hydrogen bond with a carboxylic acid residue of the channel protein. The ma rkedly low affinity of TTX-8-O-hemisuccinate may possibly be ascribable to intramolecular salt-bridge formation, which neutralizes the positive charge of the guanidinium group.