© 1995 American Heart Association, Inc.
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High- and Low-Affinity Sites for [3H]Dofetilide Binding to Guinea Pig Myocytes
From the Cardiovascular Research Group, The University of Calgary (Canada).
Correspondence to Dr Henry J. Duff, Department of Medicine, The University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada.
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±0.3x10-8 mol/L; Bmax, 76±15 fmol/106 myocytes) and a low-affinity site (Kd, 1.64±0.4x10-6 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±0.8x10-7 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.
Key Words: [3H]dofetilide • delayed rectifier K+ current • ventricular myocytes
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