ACTIVATION KINETICS OF RECOMBINANT MOUSE NICOTINIC ACETYLCHOLINE-RECEPTORS - MUTATIONS OF ALPHA-SUBUNIT TYROSINE-190 AFFECT BOTH BINDING AND GATING

Affinity labeling and mutagenesis studies have demonstrated that the c onserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subu nit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutatio ns at Y190. In Y190F the dissociation constant for ACh binding to clos ed channels was reduced similar to 35-fold at the first binding site a nd only similar to 2-fold at the second site. At both binding sites th e association and dissociation rate constants were decreased by the mu tation. Compared with wildtype AChRs, doubly liganded alpha Y1S0F rece ptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully oc cupied-open), there is an increase of similar to 6.4 kcal/mol caused b y the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes fr om altered agonist binding to the first and second binding sites, resp ectively. The closing rate constant of alpha Y190F receptors was the s ame with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was similar to 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel bl ock by ACh was similar to 2-fold lower in alpha Y190F receptors compar ed with in wildtype receptors, suggesting that there are changes in th e pore region of the receptor as a consequence of the mutation. The ac tivation reaction is discussed with regard to energy provided by agoni st-receptor binding contacts, and by the intrinsic folding energy of t he receptor.