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

Dou, Ying; Goodchild, Samuel J.; Velde, Robert Vander; Wu, Yue; Fedida, David

doi:10.1152/ajpcell.00147.2013

Cited by 6 publications

(9 citation statements)

References 37 publications

(43 reference statements)

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“…As an alternative, we used methanethiosulfonate (MTSET) to react with and study the exposure and subsequent ion current modification of a cysteine located in the upper S4 helix. For this purpose, a Cys was introduced in position 521 to follow its modification in the presence of MTSET [ 14 , 61 ], and two endogenous Cys were mutated (C445V and C449V in the S1–S2 linker [ 14 , 15 ]) to prevent any inadvertent effect caused by their modification by MTSET. As previously reported [ 14 , 15 ], very little effects on gating were induced by these mutations (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It is important to emphasize that the relatively slow time course of MTSET effects in both the continuous and different split channels with the I521C mutation is not due to any constraints imposed by our measuring system. Thus, (i) collapse of the cell membrane potential by a high K + extracellular OR-2 solution was completed in a few seconds, and (ii) the time constants of MTSET effects during the test ramps were around 30% smaller in continuous channels carrying a more extracellularly exposed cysteine at position 520 instead of 521 [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

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Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

Peña

Domı́nguez

Barros

2017

Pflugers Arch - Eur J Physiol

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Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4–S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4–S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4–S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4–S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.Electronic supplementary materialThe online version of this article (10.1007/s00424-017-2093-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

Peña

Domı́nguez

Barros

2017

Pflugers Arch - Eur J Physiol

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“…2 (gating currents). The C-less I521C mutant exposed to various thiol-modifying reagents has previously been characterized (27).…”

Section: Molecular Biologymentioning

confidence: 99%

“…Xenopus laevis oocytes were obtained and defolliculated as described previously (9). Selected stage V-VI oocytes were injected using a microdispenser (Drummond Scientific, Broomall, PA) with 50-100 ng of cRNA and incubated for 24-72 h before experiments (9,27).…”

Section: Xenopus Oocyte Preparation and Expressionmentioning

confidence: 99%

“…We have previously shown that I521C, close to the top of S4, exhibits clear state-dependent effects upon exposure to MTSET. It shows little response to MTSET at À120 mV but its deactivation kinetics are modified in a voltage-dependent manner upon depolarization (27,31). The experimental method and tail current modification are illustrated in Fig.…”

Section: Mtset Modification Experiments Confirm Voltage-dependent S4 mentioning

confidence: 99%

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The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues

Dou

Macdonald

et al. 2017

Biophysical Journal

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Kv11.1 (hERG) is a voltage-gated potassium channel that shows very slow ionic current activation kinetics, and an unusual underlying biphasic gating charge movement with fast and slow components that differ greatly in time course. The structural basis and role of the fast component of gating charge (Q) is unclear, and its relationship to the slow activation of hERG channels is not understood. In this study we have used the cut-open oocyte voltage-clamp technique to investigate the relationship of fast gating charge movement-to-residue interactions between D411 at the bottom of the S1, and lower S4 domain charged and uncharged residues. Neutralization of D411 or K538 and V535A prevented Q and greatly accelerated overall charge movement. Voltage-clamp fluorometry showed a loss of a fast component of S4 fluorescence in D411N, V535A, and K538Q upon depolarization, whereas [2-(trimethyl ammonium) ethyl] methanethiosulfonate chloride modification of I521C in the outer S4 was enhanced at more negative potentials and at earlier times in these same mutants. A functional interaction between these regions during activation was suggested by ΔΔG values >4.2 kJ/mol obtained from double mutant cycle analysis. The data indicate that interactions of S1 residue D411 with lower S4 residues stabilizes early closed states of the channel, and that disruption of these interactions results in both faster rates of activation gating and an elimination of the fast component of gating charge movement and of fluorescence. We propose that the Q charge movement during activation accompanies transitions through early closed states of the hERG activation pathway, and that the weak voltage dependence of these transitions limits the overall activation rate of hERG channels. Disruption of the D411-S4 interactions destabilizes these early closed states, leaving hERG channels able to activate at a rate similar to conventional potassium channels.

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Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain

Peña

Domı́nguez

Barros

2018

Pflugers Arch - Eur J Physiol

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Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4–S5 linker level, but also those split at the intracellular S2–S3 and the extracellular S3–S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2–S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3–S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4–S5 linker, structural integrity of the intracellular S2–S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3–S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.Electronic supplementary materialThe online version of this article (10.1007/s00424-018-2135-y) contains supplementary material, which is available to authorized users.

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

Cited by 6 publications

References 37 publications

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues

Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain

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