1. Consolidated peer review report (18 September 2021)

    GENERAL ASSESSMENT

    Lipids are known to modulate the activity of many different ion channels, with different functional outcomes depending on type of lipid, ion channel subtype, and site and mechanism of action. Thus, lipids are important endogenous modulators of channel activity and cellular physiology, and have inspired development of pharmacological compounds that mimic lipid effects. However, the effect of lipids on several members of the extensive family of K2P channels, involved in regulating cellular excitability and hormone secretion, as examples, remains relatively unstudied. The work by Riel and co-workers addresses this by studying the effect of the polyanionic lipids PIP2 and Oleoyl-CoA on 12 mammalian K2P channels expressed in Xenopus oocytes, using electrophysiology. The systematic comparison of the lipid response of different K2P subtypes allows the authors to identify which K2P subtypes that respond to PIP2 and Oleoyl-CoA and whether the lipids activate or inhibit channel activity. Interestingly, members of the TREK, TALK and THIK subfamilies are in general activated by polyanionic lipids. In contrast, members of the TASK, TWIK and TRESK subfamilies are in general inhibited by polyanionic lipids or insensitive. Moreover, the authors explore properties of the lipid and some K2P channel subtypes important for the effect, which provides indications of putative underlying mechanisms of action. The authors conclude that many members of the K2P family of ion channels are highly sensitive to polyanionic lipids, with versatile responses depending on channel subtype, and suggest that PIP2-induced inhibition of specific TASK channels is mediated through a newly identified gate in the ion-permeation pathway.

    The manuscript is clearly presented and offers a comprehensive view of how channels in the K2P family respond to PIP2 and LC-CoAs. The findings of the present study are of interest to the field and are likely to be of physiological importance. A strength of the work is the systematic comparison of the responses of a large set of K2P subtypes under similar experimental conditions, which allows the authors to draw conclusions about subtype specific responses without the caveat of comparing data collected in different experimental models and by different research groups. However, specific aspects of how experiments and analysis were performed, and the basis for how mechanistic conclusions were drawn could be better described. In the future, it will be interesting to carry out additional mechanistic studies to localize the binding sites for these lipids and to explore the physiological relevance of lipid modulation.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. It is interesting that the authors address the putative mechanistic basis for PIP2 and LC-CoA effects. However, this discussion should be extended by more clearly explaining how the authors envision the so-called X-gate to be involved in lipid effects. Do the authors believe that the L244 and R245 residues are directly involved in PIP2 binding/effects or is a downstream functional X-gate required for lipid effects in another site? Similarly, the authors should develop their hypothesis about the role of the SF-gate in lipid effects. Could these polyanionic lipids flip-flop to the extracellular leaflet of the membrane to interact with the SF-gate directly or indirectly, or are these effects likely to be mediated from the intracellular leaflet (as indicated in Figure 5)? Are there any indication of lipid effects when applied in the extracellular solution?
    2. We appreciate that the authors present their data as a condensed results text. However, the manuscript would benefit from a more extensive description of how specific experiments and analysis were performed, to facilitate for readers outside the K2P field. One of the important conclusions of this study is that different K2P subtypes respond differently to polyanionic lipids. To further consolidate this conclusion, the authors could more extensively describe how they control that effects are truly mediated by the lipid. In particular, the authors could describe how they handle experiments with current run-down under control conditions (such as examples in Figure 1D and Figure 2B for TWIK-1), as current run-down under control conditions could mask activating effects or exaggerate inhibiting effect. Moreover, the authors show some examples of control experiments with vehicle only (DMSO, like in Supplementary Figure S1I). However, it is not clear to us if control experiments with vehicle were performed systematically for all channels or if DMSO was always added to the control solution. Also, it is unclear what final concentrations of DMSO in the perfusate were achieved in the electrophysiology experiments. Please clarify these points and comment on whether any of the channels are sensitive to DMSO per se, which could complicate quantification of lipid effects. Lastly, what was the rationale behind quantifying effects at +80 mV for the data shown in Figure 3 (but at +40 mV for other data sets)? Why was RbCl, instead of KCl, used in some experiments and why was the lipid effect in those cases relative the Rb+ effect instead of the basal effect? Also, please explain the rationale of using TPA, which is listed in the methods section and used in Figure 4. Do we assume correctly that TPA is used to quantify the "unblockable current" described in the concentration-response quantification?
    3. The authors should include statistical analysis throughout their manuscript.

    Additional suggestions for the authors to consider:

    1. Abstract: Given there are K2P channels tested that do not show significant response to PIP2 (TWIK1, TRESK) or LC-CoA (TASK-1), the statement "we report strong effects of polyanionic lipids for all tested K2P channels" should be revised.
    2. Methods: A known activating mutation in hTHIK-2 is introduced to increase surface expression and macroscopic currents. It is not clear whether this activating mutation affects phospholipids response.
    3. The fact that TASK-2 belongs to the TALK, and not TASK, family may be confusing to readers outside the K2P field. Especially since TASK-2 shows a lipid response more similar to that of TASK-1 and TASK-3 than that of TALKs. To avoid confusion, the authors could make a note in the introduction (following the sentence "Members of the TALK subfamily are activated by high pH") to point out that despite the name, TASK-2 belongs to the TALK family.
    4. The results section under sub-heading "PIP2 causes subtype-dependent responses (activation/inhibition) in most K2P channel" is not easy to follow. This is mainly because the authors do not describe the results in the order the panels in Figure 1 are presented. We suggest that the authors revise the text or the figure to harmonize the order of data presentation.
    5. Page 9 - "Here we report the inhibition of TASK-2, TASK-1, TASK-3, TWIK-1 and TRESK by the polyanionic lipids PIP2 and oleoyl-CoA." - not entirely accurate as TWIK-1 and TRESK were not inhibited by PIP2, and TASK-1 was not inhibited by oleoyl-CoA. This is more accurately stated at the beginning of the Discussion.
    6. Page 10 - "In the Kir channel family all members (i.e. Kir1.x, Kir2.x, Kir3.x, Kir4.x and Kir5.x) are thought to require PIP2 as mandatory co-factor to be functional (Huang et al., 1998; Logothetis et al., 2007; Furst et al., 2014).", but this should also include Kir6.x and Kir7.x. Also, consider revising the term "mandatory", as there may be good evidence for 2.x, 3.x and 6x, but it is less clear if it is mandatory for the others.
    7. Some of the K2P channels such as TRAAK and TALK-2 show large response to PIP2 or oleoyl-CoA. Can the authors comment on the intrinsic open probability of these channels and what the magnitude of modulation one can expect from physiological changes of PIP2 and LC-CoAs?
    8. The authors cited previous work by Niemeyer et al., which showed that TASK-2 channels are activated by the short-chain PIP2 derivative dioctanoyl-PIP2. Have the authors tried dioctanoyl-PIP2 at concentrations similar to those used for long chain PIP2 and see whether TASK-2 channels are activated or inhibited in their hands?
    9. Readers not experienced in working with PIP2 might find it odd that not all of the traces shown reach a clear steady-state upon perfusion with PIP2, presumably from the accumulation of PIP2 in the membrane. It might be worth including a discussion of the difficulties of achieving an equilibrated system with full-length PIPs, and clarifying exactly how the 'stable current level' referred to in the data analysis section is defined.
    10. The authors switch between referring to fold-activation and percentage inhibition (e.g. P6, Fig 1A) - for the sake of comparison it might be simpler to choose one of these descriptors, although this is clearly a choice and up to the authors.
    11. The different effect of low (0.1 uM) and high (10 uM) PIP2 concentrations shown in Figure 1D is intriguing. This prompts the question whether the authors could provide further insights into relative PIP2 affinity of different K2P subtypes based on their control recordings. For instance, is there any particular pattern of which K2P subtypes show current run-down under control condition? A related note is whether K2P subtypes that were identified as PIP2 insensitive rather could have a high enough affinity for PIP2 to prevent PIP2 depletion and subsequent current run-down under control conditions. If so, could the lack of response of those K2P subtypes rather reflect that the PIP2 effect is already saturated?
    12. Consideration of mechanical effects of introducing PIP2 into an excised cell membrane for the mechanosensitive K2Ps (TRAAK, TREK1/2?) - even very small changes in membrane composition can have observable effects on membrane structure (e.g. Lundbaek and Andersen, 1994; Lundbaek et al. 2004; Veatch et al. 2007). Discerning between mechanosensitive effects and direct PIP2 effects does not seem straightforward given the current experiments - some discussion of this should be included.
    13. P10-11 - When discussing the fact that the breakdown of PIP2 appears to be not critical for inhibition of TASK-1/3, Lindner et al 2011 (https://physoc.onlinelibrary.wiley.com/doi/10.1113/jphysiol.2011.208983) is another reference in support.
    14. Pharmacological activation of phospholipase C as performed by applying m-3M3FBS results in more complex downstream effects than just reducing PIP2 concentrations, for example, increasing diacylglycerol concentrations as the authors cite in Wilke et al (2014). Discussion of this should be included in the sections regarding the THIK-1 experiments, indicating that the inhibition observed may not necessarily be due to PIP2 depletion.
    15. The discussion section about physiological implications: if possible, please discuss how the concentrations of lipids used in this study relates to physiological concentrations of PIP2 and LC-CoA.
    16. In the oocyte experiments, the same KCl concentration (120 mM) is used in the intracellular and extracellular solution. The activity of some voltage-gated K channels is influenced by the extracellular K+ concentration. Is this the case also for K2P channels and could this unphysiological extracellular K+ concentration impact the lipid effect?
    17. Fig 1A - the choice of a linear, broken scale for fold activation makes it difficult to agree with the authors that the fold-increase in TALK-1/2 and THIK-2 are meaningful - very hard to see the bars and error bars! Maybe the authors could consider a logarithmic scale or other alternatives? The same is true for Fig 2A.
    18. Figure 2B: The IV curves for TRAAK and TALK-2 do not seem to cross the 0 xy intercept. Can the authors explain why this is the case?
    19. The experiments presented in Figure 3 show that different LC-CoA compounds were applied on the same patch, with a BSA wash-out step in-between each application. Were these experiments always performed with the same order of compounds applied or was the effect dependent on the order of LC-CoA application? We ask this because BSA could potentially bind additional lipid components of the membrane, which could alter the baseline condition in-between LC-CoA applications.
    20. Figure 4 - It would be great to also see example traces for TASK-2 and it's mutant. One is included in Fig S1J, but it would seem to be more appropriate here.
    21. Table S3: Please comment on the different magnitude of the effect of 3 uM oleoyl-CoA determined in the different data sets (average fold activation of 4.4 and 2.3, respectively). This is a fairly large variability.

    REVIEWING TEAM

    Reviewed by:

    Sara I. Liin, Associate Professor, Linköping University, Sweden: ion channel mechanisms and lipid regulation

    Samuel Usher, PhD student at (F.M. Ashcroft lab, University of Oxford, UK): ion channel lipid regulation and fluorescence spectroscopy

    Show-Ling Shyng, Professor, Oregon Health & Science University, USA: ion channel function, regulation and structure

    Curated by:

    Stephan A. Pless, Professor, University of Copenhagen, Denmark

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

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