Dataset Information

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(+) channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (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.

SUBMITTER: Wu W

PROVIDER: S-EPMC4352586 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Concatenated hERG1 tetramers reveal stoichiometry of altered channel gating by RPR-260243.

Wu Wei W Gardner Alison A Sanguinetti Michael C MC

Molecular pharmacology 20141217 3

Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (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 preve ...[more]

PMID: 25519838

Similar Datasets

Project description:IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

Project description:ObjectiveN-methyl-d-aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1-3b[I481V]), p.Ala666Ser (GluN1-3b[A666S]), and p.Tyr668His (GluN1-3b[Y668H]).MethodsWild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy.ResultsOur data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single-receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors.SignificanceOur study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder.

Dataset Information

Concatenated hERG1 tetramers reveal stoichiometry of altered channel gating by RPR-260243.

Publications

Concatenated hERG1 tetramers reveal stoichiometry of altered channel gating by RPR-260243.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets