Evidence for multiple antiarrhythmic binding sites on the cardiac rapidly activating delayed rectifier K+ channel

We have previously shown that [3H]dofetilide binds with high affinity to sites associated with the guinea pig cardiac rapidly activating delayed rectifier K+ (IKr) channel and that class III antiarrhythmic agents, including dofetilide, clofilium, quinidine, sotalol, and sematilide, competitively displace [3H]dofetilide with IC50 values that correlate with those for blockade of the IKr channel. In this report, we show that other class III antiarrhythmic agents, namely, E‐4031 (1‐[2‐(6‐methyl‐2‐pyridyl)ethyl]‐4‐(4‐methylsulfonylamidobenzoyl)piperidine) and L‐691, 121 (3,4‐dihydro‐1′‐[2‐(benzofurazan‐5‐yl)ethyl]‐6‐methanesfulonamidospiro[(2H)‐1‐benzopyran‐2,4′‐piperidin]‐4‐one), potently block guinea pig IKr channels with respective IC50 values of 29 and 8 nM, yet have a low potency for displacement of [3H]dofetilide. Moreover, WIN 61773‐2 [(R)(+)‐4,5‐dihydro‐4‐methyl‐1‐phenyl‐3(2‐phenylethyl)‐(1H)‐2,4‐benzodiazepine monohydrochloride] biphasically displaces [3H]dofetilide according to a two site competitive binding model (site 1 = 21% displacement, IC50 = 116 nM; site 2 = 79% displacement, IC50 = 50 m̈M) with correlation to IKr block in the first phase (IC50 = 92 nM). These findings suggest that E‐‐4031, L‐691, 121, and WIN 61773‐2 inhibit IKr channels by interacting at sites distinct from the high affinity [3H]dofetilide binding site. The partial displacement of [3H]dofetilide by low concentrations of WIN 61773‐2, correlated with complete block of IKr, suggests allosteric modulation of the dofetilide binding site by this agent.