Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor

This study examines the mechanism of action of reversible antagonists of the acetylcholine receptor, focusing on the relationship between antagonist binding and inhibition of the agonist-induced permeability response. Using intact BC3H-I clonal muscle cells, measurements of competition between antagonists and ¹²⁵I-labeled α-toxin for surface receptors reveal Hill coefficients between 0.5 and 0.9 for antagonist association, showing that antagonists combine reversibly with multiple receptor sites of different affinity. Competition data are fit by a minimal model in which antagonists combine with two sites of equal number, A and B, that have affinities which can be distinguished prior to ligand association. The computed intrinsic dissociation constants, K(A) and K(B), yield selectivity ratios, K(B)/K(A), ranging between 4 for alcuronium and 89 for dimethyl-d-tubocurarine. Parallel measurements of inhibition of the initial rate of carbamylcholine-induced ²²Na⁺ influx show Hill coefficients for functional antagonism between 0.9 and 1.2. When K(B)/K(A) approaches unity, the antagonist can block the response by combining with either the A or B site. In contrast, when K(B)/K(A) is large, as with dimethyl-d-tubocurarine, the antagonist blocks the response by combining primarily with the high affinity A site. The results show the two distinct sites are confined primarily to one functional receptor oligomer. In the absence of competing ligands, α-toxin shows no preference for binding to the A or B site, but in the presence of the highly selective antagonist, dimethyl-d-tubocurarine, toxin combines preferentially with the low affinity, B site. After α-toxin combines selectively with half of the available receptor sites, residual sites not occupied by toxin have an affinity for the reversible antagonist coincident with the high affinity dissociation constant of the original two-component binding function. When bound selectively, α-toxin blocks the response more effectively than when it combines with an equal number of sites in the absence of the selective ligand. Thus, functional surface receptors contain at least two initially distinguishable binding sites, both of which must be available to the agonist to elicit a permeability response.