Activation of human ether-a-go-go-related gene 1 (hERG1) K+ channels mediates cardiac action potential repolarization. Drugs that activate hERG1 channels represent a mechanism-based approach for the treatment of long QT syndrome, a disorder of cardiac repolarization associated with ventricular arrhythmia and sudden death. Here, we characterize the mechanisms of action and the molecular determinants for binding of RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR), a recently discovered hERG1 channel activator. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured by using the two-microelectrode voltage-clamp technique. RPR induced a concentration-dependent slowing in the rate of channel deactivation and enhanced current magnitude by shifting the voltage dependence of inactivation to more positive potentials. This mechanism was confirmed by demonstrating that RPR slowed the rate of deactivation, but did not increase current magnitude of inactivation-deficient mutant channels. The effects of RPR on hERG1 kinetics and magnitude could be simulated by reducing three rate constants in a Markov model of channel gating. Point mutations of specific residues located in the S4S5 linker or cytoplasmic ends of the S5 and S6 domains greatly attenuated or ablated the effects of 3 μM RPR on deactivation (five residues), inactivation (one residue), or both gating mechanisms (four residues). These findings define a putative binding site for RPR and confirm the importance of an interaction between the S4S5 linker and the S6 domain in electromechanical coupling of voltage-gated K+ channels.
Drug-induced long QT syndrome is a disorder characterized by impaired repolarization of the ventricular action potential which can lead to arrhythmia and sudden death. The most common form involves block of hERG1a channels which encode Ikr current in myocytes. Compounds which activate hERG1a channels could then provide an effective treatment for this disorder. We have studied a recently identified hERG1a activator, PD-118057, using the two microelectrode voltage clamp technique to analyze its affect on hERG1a current expressed in Xenopus oocytes. At 10 microM, PD118057 enhanced wild-type hERG1a current by slowing the rate of inactivation and shifting the voltage dependence to more positive potentials. Consistent with an inactivation perturbing mechanism, PD118057 failed to enhance the current magnitude of an inactivation removed hERG1a mutant channel, G628C/S631C. Unlike other hERG1a activators such as RPR260243, PD118057 did not alter the rate of hERG1a deactivation, suggesting a distinct binding site on the channel protein. Wild-type dEAG1 does not exhibit C-type inactivation and, as expected was unaffected by 10 microM PD118057. A single mutation in the S6 domain (Ala478Gly) of dEAG1 introduced a fast inactivation mechanism with similar time constants as for hERG1a channels. Ala478Gly dEAG current magnitude was enhanced by PD118057 in a concentration dependent manner through removal of the fast inactivation process. In contrast, other S6 mutations that introduce a marginally slower inactivation mechanism did not exhibit sensitivity to PD118057.