Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization

Abstract Kinetic analysis of the flux of sodium ions in a heterogeneous population of acetylcholine receptor-rich microsacs (vesicles) formed by membrane fragments of electroplax indicated that functional microsacs, which on average comprise only 15% of the preparation, can be filled with 190 mM sodium chloride while nonfunctional microsacs are filled by 190 mM cesium chloride. The functional microsacs have then been successfully separated from nonfunctional microsacs on the basis of their density differences with a continuous sucrose-190 mM cesium chloride density gradient. In the presence of acetylcholine analogs all the internal sodium ions in these microsacs rapidly exchange with external ions. The efflux of sodium ions follows a single exponential decay. The isolation of functional microsacs opens up at least two new avenues of investigation of the molecular mechanism of receptor-mediated processes. The first deals with the efficiency of the process, and the second with the characterization of membrane components important in this process. The conclusions reached so far are: (i) The efficiency of the receptor-mediated process that allows inorganic ions to equilibrate across the membranes of the microsacs can adequately account for electrophysiological results obtained with muscle and nerve cells. (ii) In the receptor-rich heterogeneous population of microsacs the concentration of receptor sites in functional and nonfunctional microsacs is about the same and is therefore not the only factor determining functionality. Significant differences between functional and nonfunctional microsacs have been found so far in the concentrations of acetylcholinesterase and Na+-K+ ATPase.