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Dofetilide, a methanesulfonanilide derivative, is a potent class III antiarrhythmic drug. Like other members of this class of K+ channel blockers, the sites in the channel to which the drug binds are unknown, although high and low affinity binding has been reported in cardiomyocytes. The most sensitive K+ channel target for dofetilide seems to be IKr, the rapid component of the repolarizing delayed rectifier K+ current. However, block of other K+ channels occurs at higher concentrations and is of special interest in regard to toxicity. Recently, we have demonstrated that hIRK, a cloned inward rectifier K+ channel (IRK) isolated from human atrium and expressed heterologously in Xenopus oocytes, is blocked by dofetilide. We report the localization of a site that is critical for dofetilide block in hIRK. We used chimeric constructs between hIRK and ROMK1, a related inward rectifier that is drug resistant. Substitution of hIRK-M2, the second putative transmembrane spanning segment of IRKs, with ROMK1-M2 increased unblocking of dofetilide by 10-20-fold in hIRK. Site-directed mutagenesis further pinpointed the effects to a single hydrophobic residue (I177) in M2. A reduction in hydrophobicity by the point mutation I177C increased recovery from block > 10-fold (1.17 sec in wild-type to 0.112 sec at -80 mV at physiological K+ concentrations), leading us to suggest that hydrophobic interactions are essential for dofetilide block in hIRK. A similar mechanism may explain dofetilide block in other ion channels, including IKr.

Use of the antihistamine terfenadine has been associated with QT prolongation and torsade de pointes. One possible mechanism is blockade of cardiac potassium channels. We therefore characterized the effects of terfenadine on potassium currents recorded from isolated human cardiac myocytes. We demonstrated terfenadine block of the transient outward current and a novel, ultra-rapidly activating, delayed rectifier K+ current (IKur), which is very sensitive to 4-aminopyridine. IKur is probably produced by the protein product of Kv1.5a, a Shaker-like potassium channel cDNA cloned from human heart. We also compared terfenadine blockade of fHK (Kv1.5a) currents stably expressed in a human embryonic kidney cell line with terfenadine blockade of IKur in human atrial myocytes. Using the patch-clamp technique, we found that terfenadine produced a time-dependent reduction in Kv1.5a current that was consistent with blockade from the cytoplasmic side of the channel. The terfenadine-sensitive Kv1.5a current in human embryonic kidney cells was similar to the 4-aminopyridine-sensitive current in human atrial myocytes. In addition to blockade of the transient outward current and IKur, terfenadine at clinically relevant concentrations blocked both the rapidly and slowly activating components of the delayed rectifier in human atrial myocytes. Blockade of these K+ currents may contribute to the cardiotoxicity associated with terfenadine usage.

Verapamil is an antagonist of L-type Ca2+ channels, and part of its binding site is located in the sixth transmembrane segment (S6) in the fourth repeat of the protein. Verapamil also blocks K+ channels, which are members of the same supergene family as Ca2+ channels. We examined the effects of verapamil on a rapidly activating delayed rectifier K+ channel (designated fHK) cloned from human heart. Verapamil inhibited 86Rb+ efflux from fHK-transfected human embryonic kidney cells with an EC50 of 4.5 x 10(-5) M. Whole-cell patch-clamp experiments revealed that verapamil induced a rapid component of fHK current inactivation but was without effect on activation. The effect was concentration and voltage dependent and was attributed to open channel blockade. The apparent association and dissociation rate constants measured at +50 mV were about 1.65 x 10(5) M-1 sec-1 and 3.48 sec-1, respectively. S6 of fHK has significant homology to that portion of the verapamil binding site identified in Ca2+ channels, and S6 is thought to form part of the inner mouth of K+ channel pores. The data support a role for verapamil as a blocker of the inner pore of voltage-dependent K+ channels in human myocardium.

Use of the nonsedating antihistamine terfenadine has been associated with altered cardiac repolarization in certain clinical settings. For this reason we examined the effects of terfenadine, and its metabolites, on a rapidly activating delayed rectifier K+ channel (fHK) cloned from human heart. fHK was stably expressed in human embryonic kidney cells, and both whole-cell currents and currents from excised inside-out patches were recorded. Terfenadine (3 microM) blocked whole-cell fHK current by 72 +/- 6%. In inside-out patches, terfenadine applied to the cytoplasmic surface blocked fHK with an IC50 value of 367 nM. The main effect of terfenadine was to enhance the rate of inactivation of fHK current and thereby reduce the current at the end of a prolonged voltage-clamp pulse. The blockade displayed a weak voltage dependence, increasing at more positive potentials. The mechanism of action of terfenadine is therefore consistent with blockade of open channels. In contrast, the metabolites of terfenadine were weakly active on fHK. IC50 values for all of the metabolites tested ranged from 27-fold to 583-fold higher than that obtained for terfenadine. It is concluded that terfenadine, but not its metabolites, blocks at least one type of human cardiac K+ channel at clinically relevant concentrations and that this activity may underlie the cardiac arrhythmias that have been associated with the use of this drug.