Dynamic properties of isolated acetylcholine receptor protein: kinetics of the binding of acetylcholine and Ca ions.

Abstract

Kinetic and thermodynamic constants for elementary steps associated with the interaction of acetylcholine (AcCh) and Ca2+ with isolated AcCh receptor from Torpedo californica were determined by chemical relaxation spectrometry. Murexide was used as a Ca2+ indicator to monitor changes in Ca2+ bound to the AcCh receptor. In the presence of AcCh this technique permitted an indirect monitoring of AcCh binding, because the AcCh and the Ca2+ binding reactions were competitively coupled. A temperature-jump perturbation in the Ca2+-murexide-AcCh receptor system induced a spectrum of relaxation processes characterized by at least 3 relaxation times: .tau.1 = 5(.+-. 1) ms; .tau.2 = 35(.+-. 5) ms; and .tau.3 = 300 (.+-. 30) s. In the presence of AcCh, the Ca2+ relaxation spectrum was altered in a characteristic way. A formalism was developed to describe the normal mode relaxation times of the coupled reaction system in terms of total concentrations of both AcCh and receptor binding sites. The analysis also allowed the determination of the stoichiometry of the reactions involved or estimation of the molecular weight of the AcCh receptor. The kinetic data suggested that the reaction of AcCh receptor proceeds in at least 2 steps. The rate constant of the association of AcCh with receptor was 2.4 (.+-. 0.5) .times. 107 M-1 s-1 at 23.5.degree., 0.1 M NaCl, 50 mM Tris.cntdot.HCl, pH 8.5. Reaction schemes consistent with the present kinetic data were discussed in terms of a physicochemical model that accounts for the rapid transient conductivity changes in excitable membranes during nerve and muscle excitation.