Long-lived metastable state and hysteresis in the binding of acetylcholine to Torpedo californica acetylcholine receptor

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Abstract
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 developed very long lived metastable states, resulting in hysteresis in binding. Under conditions where the concentration of free [3H]acetylcholine is both < 0.1 .mu.M 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 it yielded the apparent equilibrium constant, .hivin.K = 5 .+-. 1 nM at 4.degree. C. The stoichiometry of the high-affinity site associated with this .hivin.K value was 1 acetylcholine/receptor monomer (MW 250,000) when carefully standardized [3H]-acetylcholine, analyzed for both radiopurity and acetylcholine concentration, was used. While 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. Therefore, the favored interpretation is either that the detergent loosens the membrane and exposes a second sterically hindered .alpha.-toxin binding site, or that it increases its affinity. The [3H]acetylcholine binding data, with solubilized and purified acetylcholine receptor predominantly in the covalently linked dimeric form (13 S), are qualitatively very similar to those with membrane-bound receptor; they indicate that the very high affinity state is induced by acetylcholine during dialysis, rather than being a preexisting conformation. The relative stoichiometry of acetycholine binding sites of purified receptor with .hivin.K = 4 .+-. 1 nM to .alpha.-bungarotoxin binding sites is close to 0.5, the same as that found in membrane fragments when the value of 125I-.alpha.-bungarotoxin binding sites assayed in the presence of nonionic detergent is used. The smallest cooperative element to account for the dialysis and rapid-mixing data is the receptor dimer, and this form is introduced in the proposed reaction scheme. The hysteresis in the acetylcholine binding to the receptor is associated with a free-energy dissipation of .DELTA.Girr = -2.8 (.+-. 0.1) kJ mol-1 and an entropy production of .DELTA.iS = 10.1 (.+-. 0.4) JK-1 mol-1, per concentration-dilution cycle at 4.degree. C. The capacity to develop long-lived metastable states may be a saving device to maintain receptors in the activatable low-affinity conformation. The hysteresis classified the receptor as a memory molecule which can reflect previous exposure to acetylcholine.