Stoichiometry of the Ligand-Binding Sites in the Acetylcholine-Receptor Oligomer from Muscle and from Electric Organ. Measurement by Affinity Alkylation with Bromoacetylcholine

Abstract

1. The affinity alkylation reaction of the cholinergic, depolarising ligand, bromoacetylcholine with reduced acetylcholine receptor in the membrane fragments of Torpedo marmorata and in Triton-solubilised receptor from cat denervated muscle has been studied.2. Brief pretreatment with 100 pM bromoacetylcholine abolishes all [3H]a-neurotoxin binding in both cases.3. In the receptor from each of these sources, the number of sites of specific a-neurotoxin binding is exactly equal to the number of sites that can be specifically alkylated by br~mol [~H]acetylcholine, at saturation of either ligand.4. The concentration-dependence of specific br~mo[~H]acetylcholine binding is found to be biphasic. A first phase can be clearly discerned in which one-half of the total specific ligand-binding sites are alkylated readily, and a second phase in which the remainder react at higher reagent concentrations. The same discrimination of two equal sets of ligand sites can be obtained by preblockade using low concentrations of unlabelled bromoacetylcholine followed by reaction with [3H]a-neurotoxin or bromo[3H]acetylcholine.5. In both phases, a single subunit of M , about 43000 is the sole site of specific alkylation in both Torpedo and muscle. The reasons for the appearance of two equal but distinct populations in the ligand binding sites in the receptors are discussed.A number of studies on the nicotinic acetylcholine receptor have endeavoured to quantify the number of binding sites for nicotinic ligands per molecule in this oligomeric protein [l -31. Measurements of the essentially irreversible binding of the specific antagonists, a-neurotoxins, to purified preparations of acetylcholine receptor, together with determination of their molecular weights, allow the number of toxin binding sites per oligomer to be estimated (for a review see [4]). However, the complicated pharmacology of the acetylcholine receptor complex [4], uncertainty in the exact receptor molecular weight, and readily reversible binding of quaternary ammonium effectors [5], are factors that have hindered the determination of the number of binding sites for Abbreviations. (cHxN)*C, dicyclohexylcarbodiimide; iPrZP-F, diisopropylfluorophosphate; T/L, stoichiometric ratio where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation of acetylcholine receptor.agonists. In practice, therefore, results have been related to a-toxin binding using a stoichiometric ratio TIL, where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation. Ligand binding in Torpedo has been investigated using the radiolabelled affinity reagent maleimido-benzyltrimethylammonium iodide [6], or more recently, bromoacetylcholine [7,8]. These reagents can specifically alkylate the acetylcholine receptor if a disulphide bond adjacent to the ligand binding site(s) has first been reduced [6,9] and studies on the Torpedo acetylcholine receptor have indicated a stoichio...

Site‐directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

“…At different times, the free sulfhydryls were reacted with an excess of bromoacetylcholine (BAC, 10 mM) and the residual c~-toxin binding sites were quantified. These experiments showed that the time-course that is required to completely reduce the disulfide bond adjacent to the ligand binding site was about 5 min in the case of ARA (20/.tM), 20 min for DTT (200/zM), in agreement with previous reports [4], and up to 2 h for MeRA or DTT at 20/tM (Fig. 3).…”

“…A well-documented model for testing this concept is the disulfide bond located between Cys-192 and Cys-193 of the a-subunit of the nicotinic acetylcholine receptor (AChoR) [1,2]. These residues are involved in the binding site of acetylcholine and have already been labeled, after reduction with DTT, with an agonist (BAC) [3][4][5][6][7] or an antagonist (MBTA) [8][9][10].…”

Pharmacological properties of immuno-isolated neuronal nicotinic receptors

“…Conservation of this disulfide bond suggests that it has an important structural or functional role in this family of proteins with a shared evolutionary history. We found no evidence that BAC labels the neuronal AChR in a biphasic manner, as is known for electric organ and skeletal muscle AChR due to preferential labeling of 1 of the 2 binding sites (Wolosin et al, 1980). Similarly, the inhibition of 3H-nicotine binding to neuronal AChR by curare (Fig.…”

“…This feature is conserved in electric organ and skeletal muscle AChRs (Damle et al, 1978;Wolosin et al, 1980), in the brain aBGT binding protein (Kemp et al, 1985;Norman et al, 1982), as well as in neuronal AChRs. Conservation of this disulfide bond suggests that it has an important structural or functional role in this family of proteins with a shared evolutionary history.…”

“…It has been found that BAC is more reactive with 1 of the 2 acetylcholine binding sites of electric organ and muscle AChR (Wolosin et al, 1980). On chicken ciliary ganglion cells BAC irreversibly inhibits activation of the AChR only after reduction with DTT (Stollberg et al, 1984).…”