PUFA stabilizes a conductive state of the selectivity filter in IKs channels
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Curated by eLife
eLife assessment
This study makes a valuable finding, a polyunsaturated fatty acid increases the conductance of a K+ channel by helping its K+ selectivity filter form a conductive state. Overall, support for this major claim is solid, though other claims remain speculative with incomplete support. These findings are expected to be of interest to researchers studying ion channel gating.
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Abstract
In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause Long QT Syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain (VSD) – which shifts the voltage dependence to more negative voltages– and the other in the pore domain (PD) – which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.
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eLife assessment
This study makes a valuable finding, a polyunsaturated fatty acid increases the conductance of a K+ channel by helping its K+ selectivity filter form a conductive state. Overall, support for this major claim is solid, though other claims remain speculative with incomplete support. These findings are expected to be of interest to researchers studying ion channel gating.
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Reviewer #1 (Public Review):
This study makes an interesting finding: a polyunsaturated fatty acid, Lin-Glycine, increases the conductance of KCNQ1/KCNE1 channels by stabilizing a state of the selectivity filter that allows K+ conduction. The stabilization of a conducting state appears well supported by single-channel analysis, though some method details are missing. The linkage to PUFA action through the selectivity filter is supported by the disruption of PUFA effects by mutation of residues which change conformation in two KCNQ1 structures from the literature. Claims about differences in Lin-Glycine binding to these two structural conformations seem to lack clear support, thus the claim seems speculative that PUFAs increase Gmax by binding to a crevice in the pore domain. A potentially definitive functional experiment is conducted by …
Reviewer #1 (Public Review):
This study makes an interesting finding: a polyunsaturated fatty acid, Lin-Glycine, increases the conductance of KCNQ1/KCNE1 channels by stabilizing a state of the selectivity filter that allows K+ conduction. The stabilization of a conducting state appears well supported by single-channel analysis, though some method details are missing. The linkage to PUFA action through the selectivity filter is supported by the disruption of PUFA effects by mutation of residues which change conformation in two KCNQ1 structures from the literature. Claims about differences in Lin-Glycine binding to these two structural conformations seem to lack clear support, thus the claim seems speculative that PUFAs increase Gmax by binding to a crevice in the pore domain. A potentially definitive functional experiment is conducted by single-channel recordings with selectivity filter domain mutation Y315F which ablates the Lin-Glycine effect on Gmax. However, this appears to be an n=1 experiment. Overall, the major claim of the abstract is supported: "... that the selectivity filter in KCNQ1 is normally unstable ... and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state." However, the claim in the abstract that selectivity filter instability "explains the low open probability" seems too general.
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Reviewer #2 (Public Review):
Summary:
Golluscio et al. address one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by polyunsaturated fatty acids (PUFA). PUFA is known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.
Strengths:
They mainly used single-channel recordings and directly assessed the behavior of the selectivity filter. The …Reviewer #2 (Public Review):
Summary:
Golluscio et al. address one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by polyunsaturated fatty acids (PUFA). PUFA is known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.
Strengths:
They mainly used single-channel recordings and directly assessed the behavior of the selectivity filter. The method is straightforward and convincing enough to support their claims.Weaknesses:
The structural model they used is the KCNQ1 channel without KCNE1 because KCNQ1/KCNE1 channel complex is not available yet. As the binding site of PUFAs might overlap with KCNE1, it is not very clear how PUFA binds to the KCNQ1 channel in the presence of KCNE1.Using other previous PUFA-related KCNQ1 mutants will strengthen their conclusions. For example, the Gmax of the K326E mutant is reduced by PUFA binding. Examining whether K326E shows reduced numbers of non-empty sweeps in the single-channel recordings will be a good addition.
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Reviewer #3 (Public Review):
Summary:
This manuscript reveals an important mechanism of KCNQ1/IKs channel gating such that the open state of the pore is unstable and undergoes intermittent closed and open conformations. PUFA enhances the maximum open probability of IKs by binding to a crevice adjacent to the pore and stabilizing the open conformation. This mechanism is supported by convincing single-channel recordings that show empty and open channel traces and the ratio of such traces is affected by PUFA. In addition, mutations of the pore residues alter PUFA effects, convincingly supporting that PUFA alters the interactions among these pore residues.
Strengths:
The data are of high quality and the description is clear.Weaknesses:
Some comments about the presentation.(1) The structural illustrations in this manuscript in general need …
Reviewer #3 (Public Review):
Summary:
This manuscript reveals an important mechanism of KCNQ1/IKs channel gating such that the open state of the pore is unstable and undergoes intermittent closed and open conformations. PUFA enhances the maximum open probability of IKs by binding to a crevice adjacent to the pore and stabilizing the open conformation. This mechanism is supported by convincing single-channel recordings that show empty and open channel traces and the ratio of such traces is affected by PUFA. In addition, mutations of the pore residues alter PUFA effects, convincingly supporting that PUFA alters the interactions among these pore residues.
Strengths:
The data are of high quality and the description is clear.Weaknesses:
Some comments about the presentation.(1) The structural illustrations in this manuscript in general need to be more clarified.
(2) The manuscript heavily relies on the comparison between the S4-down and S4-up structures (Figures 3, 4, and 7) to illustrate the difference between the extracellular side of the pore and to lead to the hypothesis of open-state stability being affected by PUFA. This may mislead the readers to think that the closed conformation of the channel in the up-state is the same as that in the down-state.
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