Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by torpedo postsynaptic membranes: stoichiometry and ligand interactions

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Abstract
Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by 2 affinities: Kdl = 33 .+-. 6 nM and Kd2 = 7.7 .+-. 4.6 .mu.M, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess .alpha.-bungarotoxin or 100 .mu.M nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCh receptors. dTC competitively inhibits the binding of [3H]AcCh and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis .alpha.-toxin in binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and .alpha.-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 .+-. 0.1:0.9 .+-. 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC, since other ligands inhibited [3H]AcCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (KI1 = 2 .mu.M, KI2 = 1 mM); Me2dTC (KI1 = 500 nM, KI2 = 10 .mu.M); decamethonium (KI1 = 100 nM, KI2 = 1.6 .mu.M). Carbamylcholine inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H]AcCh cannot be completely accounted for by competitive interaction with 2 different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H]AcCh binding function itself. dTC binds only to the AcCh sites in Torpedo membranes and that those sites display 2 affinities for dTC but only 1 for AcCh.