Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Elliott, David; Döndaş, Naciye Yaktubay; Munsey, Tim S.; Sivaprasadarao, Asipu

doi:10.3109/09687680903321081

Cited by 20 publications

(24 citation statements)

References 34 publications

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“…Together, these data suggest that the fast initial fluorescence quenching from the labelled E519C:C445V:C449V channel may provide an accurate report of a rapid environmental change at the top end of S4 in hERG channels that signals channel activation. These results are consistent with recent work using the thiol modifying reagent pCMBS which showed that the extent of exposure of S4 in hERG during depolarization is similar to other Kv channels and KvAP [32].…”

Section: Discussionsupporting

confidence: 92%

“…Fluorescence quenching accords with TMRM movement to a more hydrophilic environment, and thus the fast fluorescence quenching we have described from E519C labelling (Figs. 5– 7) may be tracking voltage sensor movement outward, as was suggested in other studies using MTSET accessibility [31] or pCMBS [32]. In Shaker A359C, and in Kv1.5 channels at A379C, voltage-dependent fluorescence quenching is predominantly fast and monotonic upon depolarization and the F-V relationship overlays [17] or approximates [12] the Q-V relationship, which is approximately 20 mV hyperpolarized with respect to the G-V relationship.…”

Section: Discussionmentioning

confidence: 55%

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Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

et al. 2010

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BackgroundhERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening.Methods and FindingsTetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V½ of activation to −27.5±2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with V½ ,1 = −37.8±1.7 mV, and V½ ,2 = 43.5±7.9 mV. The first phase, V½ ,1, was ∼20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V½ = −18.3±1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V½ = −34.4±1.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V½ = −20.6±1.2, k = 11.4 mV) and L520C quenching during depolarization (V½ = −26.8±1.0, k = 13.3 mV) matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from −40 to −110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing.ConclusionThe data indicate: 1) that rapid environmental changes occur at the outer end of S4 in hERG channels that underlie channel activation gating, and 2) that secondary slower changes reflect channel pore opening during sustained depolarizations, and channel closing upon repolarization. 3) No direct evidence was obtained of conformational changes related to inactivation from fluorophores attached at the outer end of S4.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 55%

Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

et al. 2010

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“…8B). Residues 526 to 528 are not exposed to extracellular MTSET upon depolarization, which is consistent with previous studies by Zhang et al (37), but are accessible to pCMBS (7,35). I521C was the residue with gating most dramatically affected by MTSET (Fig.…”

Section: Discussionsupporting

confidence: 90%

“…This analysis is supported by the modeling of Lee et al (10). An alternative view is that Shaker R1 aligns with hERG K1, and this is supported by S4 mutational studies (17) and the accessibility of the outer S4 region to pCMBS with depolarization (7). The outer four S4 positive charges in Shaker carry most of the activation gating charge (1,3,25), and mutation of the outer three charges in hERG to cysteines affects gating charge transfer the most (37).…”

mentioning

confidence: 94%

The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium

Dou

Goodchild

Velde

et al. 2013

American Journal of Physiology-Cell Physiology

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The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1-S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage (G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520-528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.

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“…Effects of RTC-1 on the hERG channel currents were determined by the two-electrode voltage clamp technique as described previously (Elliott et al 2009 2) using microelectrodes made from borosilicate glass, filled with 3 M KCl, which had resistances between 0.5 and 2.0 M. The effects of compounds on tail currents at −50 mV were examined using a repeated pulse protocol. For this, control currents were first measured in Ringer's solution during repeated depolarizing steps (+ 30 mV) delivered from a holding potential of −80 mV.…”

Section: Effects Of Rtc-1 On the Herg Channelmentioning

confidence: 99%

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice

Martin¹,

Leonard²,

Devine³

et al. 2016

Journal of Molecular Endocrinology

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Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL−1), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

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Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Cited by 20 publications

References 34 publications

Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice

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