Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

Thouta, Samrat; Sokolov, S. D.; Abe, Yuki; Clark, Sheldon; Cheng, Yen May; Claydon, Tom W.

doi:10.1016/j.bpj.2014.01.035

Cited by 35 publications

(39 citation statements)

References 49 publications

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“…Data were fitted to a double exponential function (n ¼ 6, 5, and 4, respectively). step depolarization to þ60 mV, which highlights the complex kinetics associated with charge transit across the membrane as has been reported previously in these channels (11,(28)(29)(30)(31). Both on-and off-gating currents display pronounced fast and slow phases of decay.…”

Section: Resultssupporting

confidence: 73%

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

Thouta

Hull

Shi

et al. 2017

Biophysical Journal

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Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (t fast ¼ 34 ms, t slow ¼ 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (D2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the ''true'' mode-shift to be~15 mV. Interestingly, the ''true'' mode-shift of gating currents was~40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

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Section: Resultssupporting

confidence: 73%

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

Thouta

Hull

Shi

et al. 2017

Biophysical Journal

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“…The alignment was obtained using Cobalt (43). In red are represented the basic residues, in yellow acidic residues, and in purple the position of the narrowest part of the bundle crossing, also the gating residue (44). The color boxes represent the transmembrane segments.…”

Section: As In Herg Covalent Binding Of S4-s5 L To S6 T Locks the K mentioning

confidence: 99%

Voltage-dependent activation in EAG channels follows a ligand-receptor rather than a mechanical-lever mechanism

Malak

Gluhov

Grizel

et al. 2019

Journal of Biological Chemistry

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Edited by Roger J. ColbranEther-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, K V 10.1 and human ether-a-go-go-related gene (hERG or K V 11.1), have revealed an original nondomainswapped structure, suggesting that the mechanism of voltagedependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5 L ) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6 T ) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5 L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in K V 10.2 channels. Using cysteine bridges and S4-S5 L -mimicking peptides, we show that the ligand/receptor model is conserved in K V 10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in K V 10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.Voltage-gated potassium (K V ) channels regulate a variety of cellular processes, including membrane polarization (1, 2), apoptosis (3), cell proliferation (4), and cell volume (5). In connection with such a variety of functions of K V channels, mutations in these channels cause a variety of pathological conditions in humans: neurological disorders (6, 7), cardiac arrhythmias (2), multiple sclerosis (8), and pain syndrome (9). It has also been shown that K V channels are associated with the development of malignant tumors cancer (10). K V 10 channels belong to the ether-a-go-go family (EAG), 5 as hERG channels. Two isoforms of K V 10 channels are expressed in mammals: K V 10.1 (eag1) and K V 10.2 (eag2), which show 70% amino acid sequence identity. K V 10.1 has been detected mainly in the brain, whereas K V 10.2 is also expressed in other tissues such as the skeletal muscles, heart, placenta, lungs, and liver (11).Atomic structures of rat K V 10.1 (12) and human hERG (K V 11.1) channels (13) highlighted many structural similarities: nonswapped pore and voltage domains, i.e. facing voltage-sensor and pore domains are from the same subunit, short S4-S5 linkers (S4-S5 L ) and similar N-terminal structures such as Per-Arnt-Sim (PAS) and N-Cap domains. We hypothesi...

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“…1B). Also like other Kv channels, KCNH channels are gated by changes in membrane potential, which cause conformational changes in the voltage sensor (S1–S4 domain) that are translated to opening of the conducting pore (lower S6) 8; 9; 10; 11 . In particular, members of the KCNH family possess N-terminal and C-terminal cytosolic regions that are unique among Kv channels 1; 2; 12 .…”

Section: Architecture Of Kcnh Channelsmentioning

confidence: 99%

The Enigmatic Cytoplasmic Regions of KCNH Channels

Morais-Cabral

Robertson

2015

Journal of Molecular Biology

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KCNH channels are expressed across a vast phylogenetic and evolutionary spectrum. In humans they function in a wide range of tissues and serve as biomarkers and targets for diseases such as cancer and cardiac arrhythmias. These channels share a general architecture with other voltage-gated ion channels but are distinguished by the presence of an N-terminal Per-Arnt-Sim (PAS) domain and a C-terminal domain with homology to cyclic nucleotide binding domains (referred to as the CNBh domain). Cytosolic regions outside these domains show little conservation between KCNH families but within a family are strongly conserved across species, likely reflecting variability that confers specificity to individual channel types. PAS and CNBh domains participate in channel gating, but at least twice in evolutionary history the PAS domain has been lost, and in one family it is omitted by alternate transcription to create a distinct channel subunit. In this focused review we present current knowledge of the structure and function of these cytosolic regions, discuss their evolution as modular domains, and provide our perspective on the important questions moving forward.

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Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

Cited by 35 publications

References 49 publications

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

Voltage-dependent activation in EAG channels follows a ligand-receptor rather than a mechanical-lever mechanism

The Enigmatic Cytoplasmic Regions of KCNH Channels

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