EQUILIBRIUM BINDING OF [TUBOCURARINE-H-3 AND [ACETYLCHOLINE-H-3 BY TORPEDO POSTSYNAPTIC MEMBRANES - STOICHIOMETRY AND LIGAND INTERACTIONS

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 two affinities: Kd1=33 ± 6 nM and Ki2=7.7 ± 4.6 μM, with equal numbers of binding sites. Both components are completely inhibited by pretreat-ment with excess α-bungarotoxin or 100 μM nonradioactive dTC and competitively inhibited by carbamylcholine with a K,=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 l(5di methylaminonaphthalene-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 α-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and α-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[formula omitted] Carbamylcholine, however, inhibited [3H]AcCh binding with a single K1=100 nM. The observed competition between those ligands and [3H]AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H]AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh. © 1979, American Chemical Society. All rights reserved.