title: High- and low-affinity sites for [3H]dofetilide binding to guinea pig myocytes.
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 x 10(-8) mol/L; Bmax, 76 +/- 15 fmol/10(6) myocytes) and a low-affinity site (Kd, 1.64 +/- 0.4 x 10(-6) mol/L; Bmax, 1620 +/- 260 fmol/10(6) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Cardiovascular Research Group, University of Calgary, Canada.
authors.forename: H J
authors.initials: HJ
authors.lastname: Duff
authors.forename: Z P
authors.initials: ZP
authors.lastname: Feng
authors.forename: R S
authors.initials: RS
authors.lastname: Sheldon
mesh_terms: D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000378:metabolism*; D007700:Kinetics; D010627:Phenethylamines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000378:metabolism*; D014316:Tritium
chemical_list: D000889:Anti-Arrhythmia Agents; D010627:Phenethylamines; D013449:Sulfonamides; D014316:Tritium; C063533:dofetilide