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

Dofetilide specifically blocks the rapid component of the delayed rect ifier current (I-KR) at nanomolar concentrations in a saturable manner , suggesting the presence of a receptor. We characterized two [H-3]dof etilide binding sites to ventricular myocytes from adult guinea pigs b y using a conventional filter assay. Scatchard analysis revealed two b inding 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-affi nity 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 dissociat ion rate constants for [H-3]dofetilide (0.02+/-0.005 min(-1) [high-aff inity site] and 0.22+/-0.064 min(-1) [low-affinity site], n=4), althou gh the observed association rate constant is equally well fit to a sin gle- or two-site model. The ability of known I-Kr blockers to compete with [H-3]dofetilide binding to both sites was assessed. E4031, clofil ium, quinidine, and sotalol competed for binding at both sites. Disopy ramide and NAPA only competed for a single binding site. The mean IC50 values for inhibition of binding to both the high- and low-affinity b inding sites correlated with their concentrations required to inhibit I-Kr in electrophysiological studies. However, inhibition of [H-3]dofe tilide binding to the high-affinity site by class III antiarrhythmic d rugs occurred at pharmacological concentrations, whereas suprapharmaco logical concentrations were required to inhibit binding to the low-aff inity site. To demonstrate that the low-affinity site was not associat ed with I-Kr [H-3]dofetilide binding was assessed in rat ventricular m yocytes, which do not express I-Kr electrophysiologically. In the rat, we observed specific binding of [H-3]dofetilide only to the low-affin ity site (K-d, 2.9+/-0.8x10(-7) mol/L; B-max, 248+/-75 fmol/10(6) myoc ytes; n=9). In addition, low concentrations of solatol (50 mu mol/L) i nhibit [H-3]dofetilide only at its high-affinity site without affectin g its low-affinity site. Thus, it can be concluded that the high-affin ity [H-3]dofetilide binding site is associated with I-Kr in guinea pig ventricular myocytes.