MECHANISM OF ACTION OF THE REDOX AFFINITY REAGENT [(TRIMETHYLAMMONIO)METHYL] CATECHOL

The synthesis of 4- and 5-hydroxy-3-[(trimethylammonio)methyl]catechol (4- and 5-HTMC) was carried out to examine their proposed involvement as intermediates in the spontaneous redox-dependent half-of-sites inactivation of neurotoxin binding sites in the nicotinic acetylcholine receptor (nAcChR) mediated by the parent compound 3- [(trimethylammonio)methyl] catechol (TMC) [Nickoloff et al. ( 1985) Biochemistry 24, 999-1007]. Oxidation of 4- and 5-HTMC occurred with sodium periodate with facile conversion to the corresponding p-quinones which were intercepted with thiols and cyclopentadiene. Both 4- and 5-HTMC inactivated neurotoxin binding in the nAcChR in a time course and over a concentration range consistent with their involvement as intermediates in the TMC redox-dependent inactivation of neurotoxin ([I-125]alpha-bungarotoxin) binding sites. Rapid concentration-dependent inactivation of neurotoxin sites occurred over a 10-1000 mu M range and was resistant to further inactivation after 50% loss of available toxin binding sites on the nAcChR. Both 4- and 5-HTMC inactivated nAcChR neurotoxin sites much more rapidly and efficiently than was observed previously with TMC. The apparent binding constants for 4- and 5-HTMC with the nAcChR, calculated from their concentration-dependent inactivation behavior toward toxin binding sites, were K-d = 224 +/- 98 and 39 +/- 17 mu M, respectively. The observed results and the redox potentials (vs Ag/AgCl reference electrode) measured by cyclic voltammetry at pH 1.8 for TMC (719 mV) and the 4- and 5-HTMC derivatives (519 and 443 mV, respectively) supported the previously proposed mechanism for inactivation of the nAcChR by TMC. Spectroelectrochemical studies of TMC and 4- and 5-HTMC under aerobic conditions confirmed the rapid and pH-dependent reversible formation of o- and p-quinones at appropriately low applied potentials. These mechanistic studies provide a basis for the design of additional redox-dependent affinity agents directed to other macromolecular sites.