Ligand-induced accelerated dissociation of (+)-cis-diltiazem from L-type Ca2+ channels is simply explained by competition for individual attachment points.

We have studied the kinetics of (+)-[3H]cis-diltiazem binding to L-type Ca2+ channels. The association reaction was of the second order with a rate constant of k1 = 5.3 x 10(-5) nM-1 min-1. A dissociation rate of the first order (k-1 = 0.0022 min-1) and the monophasic equilibrium binding curve (KD = 42 nM) seemed to reflect the simple case of one ligand binding to one site. However, when dissociation was monitored after isotopic rather than volume dilution, the dissociation rate constant increased with the concentration of added non-radioactive (+)-cis-diltiazem. Half-logarithmic plots were linear with rate constants of 0.0058, 0.011, 0.015, and 0.020 min-1 when 3, 10, 30, and 50 microM (+)-cis-diltiazem was added, respectively. We quantitatively analyzed our experimental data in terms of three different binding models, which can account for ligand-induced accelerated dissociation: 1) the classical allosteric model of pre-existing conformational states, 2) a general reaction scheme for two sites with negative cooperativity, and 3) competition of ligands for single attachment points within one binding site. Computer fitting of the experimental data to simplified reaction schemes derived from these models revealed that all three can be used to fit our data. However, only the multipoint attachment model does not require additional structural assumptions, not even an additional binding site, and is supported by structure-activity relationship analysis of diltiazem analogues. We conclude that ligand-induced accelerated dissociation should be a widely observed phenomenon that does not prove a non-competitive binding mechanism. Instead, it can be explained with the thermodynamics of non-covalent binding, where high affinity results from the cooperative interaction of a ligand molecule with multiple attachment points.