Interaction of di-iodinated 125I-labelled α-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat

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Abstract
Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibers were characterized by their interaction with di-iodinated 125I-labeled .alpha.-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22.degree. C). The time course and concentration dependence of 125I-labeled .alpha.-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labeled .alpha.-bungarotoxin (1.4-200 nM) the bimolecular-association rate constant, k+1, was (2.27 .+-. 0.49) .times. 104 M-1 .cntdot. s-1 (mean .+-. SD, n = 10). In concentration-dependence experiments, k+1 was 2.10 .times. 104 M-1 .cntdot. s-1 and 1.74 .times. 104 M-1 .cntdot. s-1 with 10 and 135 min incubations, respectively. In association experiments the 1st-order rate constant was proportional to the 125I-labeled .alpha.-bungarotoxin concentration. 125I-Labeled .alpha.-bungarotoxin dissociation was 1st order with a dissociation constant, k-1, less than or equal to 3 .times. 10-6 s-1 (half-life .gtoreq. 60 h.) A single class of high-affinity toxin-binding sites at the end-plate was indicated with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 .+-. 0.46) .times. 107 (mean .+-. SD, n = 22)/rat end-plate. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labeled .alpha.-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. Agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labeled .alpha.-bungarotoxin binding. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (.DELTA.S0 = +0.46 kJ .cntdot. mol-1 .cntdot. K-1) than the antagonist tubocurarine (.DELTA.S0 = +0.26 kJ .cntdot. mol-1 .cntdot. K-1). The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. The affinity and specificity of 125I-labeled .alpha.-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibers were the same. The study of binding only requires mg amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.