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 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...

Single-channel current recordings of acetylcholine receptors in electroplax isolated from theElectrophorus electricus main and Sachs' electric organs

“…& Nachmansohn, 1957;Schoffeniels, 1957Schoffeniels, , 1959. The Main organ occupies most of the body of the eel and has proved to be a rich source of acetylcholine receptors for studies of acetylcholine-dependent ion flux in membrane vesicles (Kasai & Changeux, 1971;Hess & Andrews, 1977;Hess et al, 1983) and of the binding of ligands to the receptor (Kasai & Changeux, 1971;Meunier & Changeux, 1973;Bu!ger et al, 1977;Fu et al, 1977). The electroplax of the Main organ are more irregular in shape and are much smaller than the cells in the organ of Sachs. Membrane vesicles prepared from the E. electricus Main organ were used to elucidate the mechanism of action of acetylcholine and other activating ligands that, upon binding to the acetylcholine receptor, induce transmembrane ion flux (Hess et al, 1979;Aoshima et al, 1980;Cash et al, 1980Pasquale et al, 1983).…”

The Effects of Ions on the Binding of Agonists and Antagonists to Muscarinic Receptors

“…anaerobiosis), and the rapid response (,-1 Ims) which is short compared with potential diffusion time of molecules within the cell. (Katz & Miledi, 1973;Hess & Andrews, 1977). On the other hand, for a number of receptors there is evidence of direct coupling to adenylate cyclase, that is, to a mechanism apparently not directly requiring an increase in ion permeability (Lefkowitz & Williams, 1978 in that the open state is able in some manner to surround the ion and provide a counterionic atmosphere, whereas in the closed state it either cannot interact at all or at any rate in a different manner.…”

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: Identification and characterization of membrane properties that affect ion flux measurements

“…(ii) About 15% of the flux comes from specific vesicles which do respond to carbamylcholine. In the presence of 1 mM carbamylcholine the exchange of inorganic ions across the vesicle membrane is complete in less than 3 sec (Hess, 1979), and in the absence of carbamylcholine in 20 hr (Hess & Andrews, 1977). In earlier experiments (Kasai & Changeux, 1971a-c) the tl/2 value for the efflux from the total vesicle population, z, has been used to assess receptor-controlled fluxes.…”

“…About 85% of the total observed efflux is due to unspecific vesicles (Hess et al, 1975). The specific vesicles have been physically separated from the unspecific ones by taking advantage of their permeability differences (Hess & Andrews, 1977).…”