HIGH- AND LOW-AFFINITY SITES FOR [H-3] DOFETILIDE BINDING TO GUINEA-PIG MYOCYTES

Dofetilide specifically blocks the rapid component of the delayed rectifier current (I-KR) at nanomolar concentrations in a saturable manner, suggesting the presence of a receptor. We characterized two [H-3]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 (K-d, 2.8+/-0.3x10(-8) mol/L; B-max 76+/-15 fmol/10(6) myocytes) and a low-affinity site (K-d, 1.64+/-0.4 x10(-6) mol/L; B-max 1620+/-260 fmol/10(6) myocytes) (n=11). Kinetic studies showed that there were two dissociation rate constants for [H-3]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 I-Kr blockers to compete with [H-3]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 I-Kr in electrophysiological studies. However, inhibition of [H-3]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 I-Kr [H-3]dofetilide binding was assessed in rat ventricular myocytes, which do not express I-Kr electrophysiologically. In the rat, we observed specific binding of [H-3]dofetilide only to the low-affinity site (K-d, 2.9+/-0.8x10(-7) mol/L; B-max, 248+/-75 fmol/10(6) myocytes; n=9). In addition, low concentrations of solatol (50 mu mol/L) inhibit [H-3]dofetilide only at its high-affinity site without affecting its low-affinity site. Thus, it can be concluded that the high-affinity [H-3]dofetilide binding site is associated with I-Kr in guinea pig ventricular myocytes.