Abstract
In earlier studies with the acetylcholine receptor (AcChoR) of Electrophorus electricus the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. Torpedo vesicles on a weight basis may contain several hundred times more receptor sites than do E. electricus vesicles. Techniques have been developed to (i) correct for the kinetic heterogeneity of the vesicle population; (ii) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted J(A), the influx rate coefficient before the onset of inactivation, to be measured); and (iii) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the E. electricus receptor accommodates the ion translocation in T. californica vesicles. The features in common are: (i) A rapid initial flux rate [J(A)(max) for T. californica is 310 sec(-1); for E. electricus it is 7.5 sec(-1)]. These differences in flux rates are consistent with a difference in AcChoR density. (ii) A rapid inactivation process [alpha(max) for T. californica is 2 sec(-1); for E. electricus it is 7 sec(-1)]. (iii) A slow AcChoR-controlled flux that continues after the rapid inactivation [J(I)(max) for T. californica is 1.3 sec(-1); for E. electricus it is 0.015 sec(-1)]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, beta, of 0.12 sec(-1). The second process leads to undetectable flux activity during the time of observation (30 sec in 10 mM carbamoylcholine). These studies are also significant because fundamental differences may exist between the mechanism of AcChoR-controlled ion flux in synaptic (Torpedo) and conducting (E. electricus) membranes.