KINETIC CONSTANTS OF THE ACETYLCHOLINE (ACH) RECEPTOR REACTION DEDUCED FROM THE RISE IN OPEN PROBABILITY AFTER STEPS IN ACH CONCENTRATION

Outside-out patches of enzymatically dissociated adult and denervated mouse muscle fibers were superfused repetitively by pulses of acetylcholine (ACh) containing solution. Up to 300 channels opened simultaneously 300-mu-s after the beginning of a 1,000-mu-M ACh pulse corresponding to a peak current l of almost -1 nA. Single responses to ACh were averaged and the concentration dependence of l and of the rise time tr from 0.1 l to 0.9 l was measured, In adult receptors, l increased proportional to the second to third power of ACh concentration, whereas in embryonic-type receptors it was proportional to the first to the second power. t(r) increased from approximately 0.3 ms at 1,000-mu-M ACh to a plateau value of approximately 5 ms for adult and of approximately 10 ms for embryoniclike receptors at concentrations < 10-mu-M ACh. The concentration dependence of l and t(r) was simulated using the standard model of ACh binding with different combinations of rate constants and two and three binding sites for ACh. The calculated curves were compared to the measurements and a set of well fitting rate constants was determined for adult and embryoniclike receptors. Three binding sites for ACh were necessary to fit the dose response for l for adult receptors. A method for deriving rate constants in a model of ACh-receptor interaction is described that avoids analysis of open-closed kinetics of single channels. which in rapid systems, as the ones studied here, are at the limit of the frequency response of the current measurement.