Stoichiometry of altered hERG1 channel gating by small molecule activators

2014

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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.

Section: Resultsmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

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

2014

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“…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 ).…”

Section: Resultsmentioning

confidence: 99%

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

2015

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Block of human ether-à-go-go-related gene 1 (hERG1) K 1 channels by many drugs delays cardiac repolarization, prolongs QT interval, and is associated with an increased risk of cardiac arrhythmia. Preferential block of hERG1 channels in an inactivated state has been assumed because inactivation deficient mutant channels can exhibit dramatically reduced drug sensitivity. Here we reexamine the link between inactivation gating and potency of channel block using concatenated hERG1 tetramers containing a variable number (0-4) of subunits harboring a point mutation (S620T or S631A) that disrupts inactivation. Concatenated hERG1 tetramers containing four wild-type subunits exhibited high-affinity block by cisapride, dofetilide, and MK-499, similar to wild-type channels formed from hERG1 monomers. A single S620T subunit within a tetramer was sufficient to fully disrupt inactivation gating, whereas S631A suppressed inactivation as a graded function of the number of mutant subunits present in a concatenated tetramer. Drug potency was positively correlated to the number of S620T subunits contained within a tetramer but unrelated to mutation-induced disruption of channel inactivation. Introduction of a second point mutation (Y652W) into S620T hERG1 partially rescued drug sensitivity. The potency of cisapride was not altered for tetramers containing 0 to 3 S631A subunits, whereas the potency of dofetilide was a graded function of the number of S631A subunits contained within a tetramer. Together these findings indicate that S620T or S631A substitutions can allosterically disrupt drug binding by a mechanism that is independent of their effects on inactivation gating.

“…Second, poor expression of mutant ERG2 channels coupled with interference from endogenous currents at negative potentials in oocytes prevented analysis of deactivation kinetics over a broad range of voltages. Third, a Hill coefficient .1 for the [RPR]-response relationships could indicate positive ligand-binding cooperativity or could result from cooperative subunit interactions, as we previously found for two other hERG1 activators, PD-118057 [2-[[4-[2-(3,4-dichlorophenyl) In our previous study of the effects of PD-118057 and ICA-105574 on concatenated hERG1 tetramers (Wu et al, 2014), we found that although subunit cooperativity contributes to the effects of these two hERG1 agonists, there was no change in EC 50 or the Hill coefficient as the number of drug binding sites was increased from one to four. This finding indicates that occupancy of one binding site did not lead to an enhancement of PD-118057 and ICA-105574 affinity of other sites.…”

Section: Rpr Has Differential Effects On Herg1 and Rerg2mentioning

confidence: 92%

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

2015

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Compounds can activate human ether-à-go-go-related gene 1 (hERG1) channels by several different mechanisms, including a slowing of deactivation, an increase in single channel open probability, or a reduction in C-type inactivation. The first hERG1 activator to be discovered, RPR260243 ((3R,4R)-4-[3-(6-methoxyquinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid) (RPR) induces a pronounced, voltage-dependent slowing of hERG1 deactivation. The putative binding site for RPR, previously mapped to a hydrophobic pocket located between two adjacent subunits, is fully conserved in the closely related rat ether-à-go-gorelated gene 2 (rERG2), yet these channels are relatively insensitive to RPR. Here, we use site-directed mutagenesis and heterologous expression of channels in Xenopus oocytes to characterize the structural basis for the differential sensitivity of hERG1 and rERG2 channels to RPR. Analysis of hERG1-rERG2 chimeric channels indicated that the structural determinant of channel sensitivity to RPR was located within the cytoplasmic C-terminus. Analysis of a panel of mutant hERG1 and rERG2 channels further revealed that seven residues, five in the C-linker and two in the adjacent region of the cyclic nucleotide-binding homology domain, can fully account for the differential sensitivity of hERG1 and rERG2 channels to RPR. These findings provide further evidence that the C-linker is a key structural component of slow deactivation in ether-à-gogo-related gene channels.