High- and Low-Affinity Sites for [ 3 H]Dofetilide Binding to Guinea Pig Myocytes

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Abstract
Dofetilide specifically blocks the rapid component of the delayed rectifier current (IKr) at nanomolar concentrations in a saturable manner, suggesting the presence of a receptor. We characterized two [3H]dofetilide binding sites to ventricular myocytes from adult guinea pigs by using a conventional filter assay. Scatchard analysis revealed two binding sites with different affinities: a high-affinity site (Kd, 2.8±0.3×10−8 mol/L; Bmax, 76±15 fmol/106 myocytes) and a low-affinity site (Kd, 1.64±0.4×10−6 mol/L; Bmax, 1620±260 fmol/106 myocytes) (n=11). Kinetic studies showed that there were two dissociation rate constants for [3H]dofetilide (0.02±0.005 min−1 [high-affinity site] and 0.22±0.064 min−1 [low-affinity site], n=4), although the observed association rate constant is equally well fit to a single- or two-site model. The ability of known IKr blockers to compete with [3H]dofetilide binding to both sites was assessed. E4031, clofilium, quinidine, and sotalol competed for binding at both sites. Disopyramide and NAPA only competed for a single binding site. The mean IC50 values for inhibition of binding to both the high- and low-affinity binding sites correlated with their concentrations required to inhibit IKr in electrophysiological studies. However, inhibition of [3H]dofetilide binding to the high-affinity site by class III antiarrhythmic drugs occurred at pharmacological concentrations, whereas suprapharmacological concentrations were required to inhibit binding to the low-affinity site. To demonstrate that the low-affinity site was not associated with IKr, [3H]dofetilide binding was assessed in rat ventricular myocytes, which do not express IKr electrophysiologically. In the rat, we observed specific binding of [3H]dofetilide only to the low-affinity site (Kd, 2.9±0.8×10−7 mol/L; Bmax, 248±75 fmol/106 myocytes; n=9). In addition, low concentrations of solatol (50 μmol/L) inhibit [3H]dofetilide only at its high-affinity site without affecting its low-affinity site. Thus, it can be concluded that the high-affinity [3H]dofetilide binding site is associated with IKr in guinea pig ventricular myocytes.