Long-lived metastable states and hysteresis in the binding of acetylcholine to Torpedo california acetylcholine receptor.
Studies of the binding of [3H]acetylcholine to receptor-rich membranes of Torpedo californica electric organ under conditions that normally lead to a state of equilibrium did not give rise to equilibrium binding curves. Instead, the acetylcholine receptor was found to develop very long lived metastable states resulting in hysteresis in binding. Under conditions where the concentration of free [3H]acetylcholine is both less than 0.1 microM and smaller or comparable to the total receptor concentration, the degree of binding of acetylcholine depends on the rate, i.e., the mode, of increasing the acetylcholine concentration (rapid mixing vs. dialysis). The equilibrium positive cooperativity in high-affinity acetylcholine binding previously inferred from the data is deceiving; the curvature in Scatchard representations is a consequence of long-lived nonequilibrium distributions between high-affinity and lower affinity receptor conformers. By manipulation of the experimental conditions, true equilibrium binding, resulting in a linear Scatchard binding curve, was obtained and yielded the apparent equilibrium constant, K = 5 +/- 1 nM at 4 degrees C. The stoichiometry of the high-affinity site associated with this K value was found to be one acetylcholine per receptor monomer (Mr 250 000) when carefully standardized [3H]acetylcholine analyzed for both radiopurity and acetylcholine concentration was used. While our fresh membrane fragments prepared in the presence of 4 mM Ca2+ revealed up to twice as many 125I-alpha-bungarotoxin sites in 0.1% nonionic detergent relative to those assayed in the absence of detergent, nonionic detergent treatment of membrane fragments did not result in any change in total available acetylcholine binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000378:metabolism; D002501:Centrifugation, Zonal; D004557:Electric Organ / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium
D002038:Bungarotoxins; D002250:Carbon Radioisotopes; D011950:Receptors, Cholinergic; D014316:Tritium; D000109:Acetylcholine