Ligand Interactions with Membrane-Bound Porcine Atrial Muscarinic Receptor(s)

Ligand interactions with the membrane-bound muscarinic acetylcholine receptor in porcine atria are characterized by using [³H]quinuclidinyl benzilate [Yamamura, H. I., & Snyder, S. H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1725-1729] as a probe for the receptor. Antagonists and local anesthetics appear to displace quinuclidinyl benzilate from a single population of high-affinity sites while agonists appear to interact with two noninterconvertible subpopulations of quinuclidinyl benzilate binding sites. The simplest mechanism that appeared to be consistent with both kinetic and thermodynamic studies of [³H]QNB binding to the receptor required two steps. The first step, in rapid preequilibrium (K = 1.8 × 10^-9 M), was followed by a slow ligand-induced conformational change (k₁ = 5.5 × 10^-3 s^-1; k_-1= 3.3 × 10^-4 s^-1) of the receptor-QNB complex. The overall dissociation constant calculated from the kinetic data (1.09 × 10^-10 M) was in good agreement with that determined from equilibrium measurements (K_m = 1.22 × 10^-10 M). The complex behavior of the dissociation rate constant for [³H]quinuclidinyl benzilate in the presence of competing ligands may indicate that at high concentrations these ligands either bind to a second low-affinity site(s) on the protein, altering the properties of the high-affinity site, or they nonspecifically alter the properties of the membrane, creating a second population of quinuclidinyl benzilate-receptor complexes that dissociate with an observed rate constant equal to ~3 × 10^-5 s^-1.