The mechanism of the acetylcholine receptor-controlled translocation of inorganic ions through membranes is being investigated. The problem is of interest because proteins like this receptor control the transmission of signals between nerve cells and nerve muscle cells. During the last 4 years we have developed methods and techniques (with a time resolution of about 5 msec) which allow kinetic measurements of ion translocation to be made in receptor-containing membrane vesicles obtained from the electric eel. The intrinsic rate constant, J, for the receptor-controlled ion translocation has been determined and ion translocation rates have been related to (i) protein isomerization rates, (ii) the equilibrium constants of the ligand binding process, and (iii) the channel opening equilibrium. The minimum model based on our measurements can be treated quantitatively and accounts for the ion translocation process over the whole range of acetylcholine (5,000x) and carbamylcholine (200x) concentration range investigated. We now plan to (i) complete our studies of receptor function in membrane vesicles in the msec time region by investigations which appear to be of physiological interest and which have been only partially resolved in studies with cells: (a) the receptor-controlled flux of Ca2+ and the effect of Ca2+ on receptor-controlled translocation of Na+ and K+, (b) the effects of transmembrane voltage, inorganic ion concentration, and temperature on the ion translocation process; (ii) develop techniques which allow chemical kinetic measurements with vesicles to be made in the M usec time region. Involved are reagents which can be photolyzed (in the M usec time region) to acetylcholine or its analogs, and involve Beta-carotene as an indicator of transmembrane voltage changes in the sub M usec region and resonance Raman spectroscopy to detect changes in membrane potential; (iii) to determine the rate constants of opening and closing of the transmembrane ion channels; (iv) extend investigations to other receptors both because of the importance of the molecules and to make the techniques which we have developed generally useful in investigations of the many known membrane-bound proteins which mediate the transfer of ions and molecules across membranes.
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