The kinetics of acetylcholine-receptor-mediated sodium efflux from electroplax microsacs of Electrophorus electricus has been analyzed. This led to the discovery that only a small fraction of the observed efflux is affected by chemical effectors such as carbamylcholine. Experimental conditions were chosen so that the receptor-mediated flux could be analyzed without the measurements' being obscured by efflux from the nonexcitable microsacs. Near equilibrium the efflux follows a single exponential decay. The apparent first order rate constant for sodium-22 efflux was determined as a function of effector concentration and is considerably higher than previously estimated. The process does not show cooperativity under the experimental conditions, in agreement with the binding isotherms of effectors and the same membrane preparation. The presence of potassium ions inhibits the receptor-mediated sodium flux. It is suggested that interaction o inorganic ions with the receptor may play an important role in the cooperative effects observed in electrophysiological experiments. Nerve impulse generation and transmission involve permeability changes of the neural membrane to Na+ and K+ (1). Initiation of increased ion flow across the membrane by the binding of chemical effectors to a specific membrane-bound receptor protein plays an important role in bioelectric phenomena (2-4). The effector-receptor interactions are believed to provide the basis for integrated neural responses. The underlying molecular mechanism, by which the interaction of chemical mediators with the receptor regulates the flux of inorganic ions, is unknown. In this paper we report the study of a fundamental process in the acetylcholine-receptor-mediated ion flux in electroplax microsacs, which is closely related to chemical mediator-receptor interaction.Electroplax membrane preparations from Electrophorus electricus were chosen for the experiments because they appeared to be uniquely suited for finding a correlation between interactions of chemical mediators with the receptor and changes of the membrane permeability to inorganic ions. Considerable insight into the chemical properties of these membranes, and the electrophysiological properties of single electroplax, comes from the work of Nachmansohn and colleagues (2-6).The acetylcholine receptor of E. electricus has been isolated in several laboratories and is being characterized (e.g., 7-11). The interaction of effectors with the receptor in electroplax microsac preparations has been studied by equilibrium and kinetic methods (e.g., 12-20,t,*). Kasai and Chan-geux have demonstrated that electroplax microsacs exhibit

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 19Q 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 reep-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.Initiation of increased ion flow across neural membranes by the combination of acetylcholine or its congeners with the membrane-bound acetylcholine receptor plays an important role in the generation (1) (11,12), the mechanism by which receptor ligands bind to the membrane-bound receptor was studied with these membrane preparations (13). We showed that there are two types of receptor binding sites, one for activators and one for inhibitors of neural excitability (12,14). Using a fluorescent lanthanide, terbium, to probe the calcium-and activatorbinding sites of the purified receptor prepared from Torpedo ocellata, we obtained additional evidence (15, 16) for different receptor binding sites for activators and inhibitors. In addition, we demonstrated a half-of-the-sites relationship between inhibitors and activators (13). On the basis of kinetic data, we developed a relatively simple two-state model for the membrane-bound receptor that takes into account both equilibrium and kinetic measurements of receptor-ligand interaction (14).Recently we investigated the kinetics of sodium-22 ion flux using the same membrane microsac preparation (17, 18). We found that sodium-22 efflux from 85% of the microsacs exhibited complex kinetics and was not affected by receptor ligands. Development of a kinetic method (17) allowed us to measure the efflux from the f...

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