Concatenated hERG1 Tetramers Reveal Stoichiometry of Altered Channel Gating by RPR-260243

Abstract

Activation of human ether-a-go-go-related gene 1 (hERG1) K 1 channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl(RPR) slows deactivation and attenuates inactivation of hERG1 channels. A detailed understanding of the molecular mechanism of hERG1 agonists such as RPR may facilitate the design of more selective and potent compounds for prevention of arrhythmia associated with abnormally prolonged ventricular repolarization. RPR binds to a hydrophobic pocket located between two adjacent hERG1 subunits, and, hence, a homotetrameric channel has four identical RPR binding sites. To investigate the stoichiometry of altered channel gating induced by RPR, we constructed and characterized tetrameric hERG1 concatemers containing a variable number of wild-type subunits and subunits containing a point mutation (L553A) that rendered the channel insensitive to RPR, ostensibly by preventing ligand binding. The slowing of deactivation by RPR was proportional to the number of wild-type subunits incorporated into a concatenated tetrameric channel, and four wild-type subunits were required to achieve maximal slowing of deactivation. In contrast, a single wild-type subunit within a concatenated tetramer was sufficient to achieve half of the maximal RPR-induced shift in the voltage dependence of hERG1 inactivation, and maximal effect was achieved in channels containing three or four wild-type subunits. Together our findings suggest that the allosteric modulation of channel gating involves distinct mechanisms of coupling between drug binding and altered deactivation and inactivation.

The Link between Inactivation and High-Affinity Block of hERG1 Channels

“…Concatenation of subunits can hERG1 Block and Inactivation affect the pharmacology of the resulting tetrameric channels (Wu et al, 2014b(Wu et al, , 2015. Therefore, we compared the concentration-response relationships for cisapride, dofetilide, and MK-499 on the block of concatenated WT hERG1 (WT 4 ) channels and channels formed naturally by coassembly of single WT subunits (WT monomer ).…”

“…Concatenated hERG1 dimers were used to show that cisapride interacts with Tyr652 residues on adjacent subunits, whereas terfenadine interacts with Tyr652 residues on diagonal, but not on adjacent, subunits (Imai et al, 2009). We previously used concatenated hERG1 tetramers to show that modification of hERG1 channel gating requires occupancy of all four available ligand binding sites to achieve maximal efficacy by the agonists PD-118057 [2-[[4-[2-(3,4-dichlorophenyl) (Wu et al, 2015). In the present study, we further explored the link between inactivation gating and potency of block by cisapride, dofetilide, and MK-499 using concatenated hERG1 tetramers containing a variable number of mutant subunits harboring the S620T or S631A mutation known to disrupt inactivation without significantly changing ion selectivity (as occurs with the double mutation G628C/S631C).…”

C-Linker Accounts for Differential Sensitivity of ERG1 and ERG2 K+ Channels to RPR260243-Induced Slow Deactivation

“…3A). The apparent reduced effect of RPR on R I tail at the less negative potentials is because I tail was measured for only 9 seconds and not at steady-state levels, which can take .30 seconds (Wu et al, 2015). The fold change in R I tail for hERG1 plotted as a function of [RPR] at five different voltages is presented in Fig.…”

“…The effects of RPR on hERG1 channel currents have been characterized in detail (Kang et al, 2005;Perry et al, 2007;Wu et al, 2015). For this study, we did not analyze the effects of RPR on the kinetics of activation or the voltage dependence of activation or inactivation.…”

“…Due to the 4-fold symmetry of voltage-gated K 1 channels, a homotetrameric hERG1 channel should contain four identical RPR binding sites. Characterization of concatenated hERG1 tetramers containing a defined number and positioning of L533A mutant subunits that prevent RPR activity (Perry et al, 2007) revealed that occupancy of all four binding sites was required to achieve the maximal slowing of deactivation by RPR (Wu et al, 2015).…”