A phosphorylation-dependent mechanism controls splice variant-specific S-palmitoylation of cardiac Kv4.3

  1. Eleanor Dickson-Murray
  2. Chunyun Du
  3. Chien-Wen S Kuo
  4. Rebecca J Gilchrist
  5. Sheon Mary
  6. Samuel Adu
  7. Outi Kämäräinen
  8. Amr Wahdan
  9. Emma Dunning
  10. Fiona Jordan
  11. Elaine Brown
  12. Jack W Houghton
  13. Daria Berezina
  14. Jianan Lu
  15. Edward W Tate
  16. Rachel C Myles
  17. Francis L Burton
  18. Godfrey L Smith
  19. Helen Walden
  20. Jules C Hancox
  21. William Fuller

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Abstract

S-palmitoylation modulates the activity of many cardiac ion channels, yet the upstream signals that control this post-translational modification (PTM) are poorly defined. Here we identify a phosphorylation-dependent mechanism that governs splice variant-specific S-palmitoylation of the potassium channel Kv4.3. Using Acyl-RAC in native tissue and heterologous cells, we map palmitoylation to C546/547 in the intrinsically disordered Kv4.3 C-terminal tail. The short Kv4.3 splice variant Kv4.3S is ∼2.5-3-fold more palmitoylated than the long splice variant Kv4.3L, and systematic mutagenesis localises the dominant inhibitory determinant of Kv4.3 S-palmitoylation to residues 488-498 within the Kv4.3L-specific splice insert. KChIP2.1 promotes accumulation of a post-translationally modified Kv4.3 species that is selectively S-palmitoylated, and nanobody-targeted dephosphorylation removes this species and reduces Kv4.3 S-palmitoylation. Phos-tag electrophoresis and C-terminal truncation mapping identify S538 as the principal phosphorylation site enabling Kv4.3 S-palmitoylation; mutation of S538 markedly reduces formation of the palmitoylation-competent species. TurboID proximity-labelling and biochemical assays indicate that phosphorylation enhances recruitment of Kv4.3 to zDHHC5. Acute kinase inhibition rapidly eliminates phosphorylation but only gradually reduces palmitoylation, revealing temporal uncoupling between these PTMs. Functionally, non-palmitoylatable Kv4.3S exhibits larger peak currents, faster inactivation, and a left-shifted activation curve, consistent with palmitoylation limiting channel function and modulating gating transitions. Together, these findings identify phosphorylation of S538 as a priming modification that licenses Kv4.3 S-palmitoylation at C546/547, explain splice-variant differences in S-palmitoylation, and define a PTM cascade that tunes Kv4.3 channel behaviour.

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    Reply to the reviewers

    Reviewer 1

    Reviewer 1, Comment 1: The authors used Acyl-RAC to assess Kv4.3 palmitoylation. Although Flot2 was included as a positive control, a negative control is missing and should be included….

    Acyl-RAC is a well-established and extensively validated method for detecting S-palmitoylated proteins. Rather than adding a Western blot for a single non-palmitoylated protein, we highlight that the most rigorous control is already provided within the study. Throughout the investigation, mutation of the two palmitoylated cysteines in Kv4.3 (C546/C547) abolishes capture of the protein by Acyl-RAC despite otherwise minimal alteration of the protein sequence. The loss of capture following removal of the palmitoylation sites provides direct evidence that Acyl-RAC capture of Kv4.3 depends on these cysteine residues and therefore reflects specific detection of palmitoylation. We consider this substrate-specific negative control to be more informative than demonstrating lack of capture of an unrelated non-palmitoylated protein. To make this point clearer for readers, we will add text to the revised manuscript highlighting that C546/C547 mutation prevents recovery of Kv4.3 in the Acyl-RAC assay and therefore serves as an internal control for the method.

    Reviewer 1, Comment 2: Statistical analysis for Fig 1J

    The scrambled mutant was assessed three times in independent replicates with identical results each time. Not every experiment included Kv4.3S or Kv4.3L, but quantitative assessment of ‘zero palmitoylation’ can be presented, if required.

    Reviewer 1, Comment 3: Doublet with KChIP2.1

    We agree that the slower-migrating Kv4.3 species is resolved most clearly in the TMD-del construct in the presence of KChIP2.1. The reviewer is correct that the doublet is also sometimes visible in experiments using full-length Kv4.3, but resolution is less consistent. We can add a comment explaining this to the text.

    KChIP2.1 was not expressed in the experiment shown in Figure 2G, which was performed using full-length Kv4.3S. We suggest the effect of LaG16-PP2A on electrophoretic mobility is very clear in this particular experiment. Comparing the unfractionated sample without nanobody (lane 1) and the unfractionated sample co-expressing LaG16-PP2A (lane 3) shows a clear loss of the upper, slower-migrating species, consistent with dephosphorylation-induced collapse of the doublet. We will revise the figure legend to help make interpretation easier.

    Since this point was also raised by Reviewer 4, we will provide an additional figure comparing only unfractionated lysates from cells transfected with YFP-Kv4.3 and the nanobodies to make the side by side comparison easier.

    We appreciate the suggestion to extend gel-running times, but we do not believe that this would alter the interpretation. The mobility shift is most robustly demonstrated using the TMD-del construct, which was specifically developed to enhance resolution of phosphorylated (slower migrating) Kv4.3 species.

    Reviewer 1, Comment 4: Rationale for focus on zDHHC5?

    We acknowledge that our data indicate zDHHCs other than zDHHC5 are capable of palmitoylating Kv4.3. We focused our studies on zDHHC5 because our primary interest is regulation of Kv4.3 in the heart, where zDHHC5 is highly expressed and has established roles in the palmitoylation and regulation of cardiac ion channels. zDHHC8 is less abundant in cardiac tissue. We will add text to the revised manuscript clarifying the rationale for prioritising zDHHC5 for detailed mechanistic investigation. We agree that a potential contribution of zDHHC8 cannot be excluded and will highlight this as an area for future study.

    Reviewer 1, Comment 5: y axis in Figure 4C?

    The reviewer is correct. We normalise co-purification of the associated protein (zDHHC5) to the amount of target protein captured (YFP-Kv4.3). We will clarify this in the figure legend.

    Reviewer 1, Minor comments:

    We will provide more background on KChIP isoforms as suggested. 2 & 3. Font sizes and labels will be corrected.

    All abbreviations will be defined. Most of the western blots in this study were performed using 4-20% polyacrylamide gradient gels cast in-house 12 at a time. During casting, small differences in hydrostatic pressure between plates in the multicasting apparatus can produce local distortions in the acrylamide gradient, resulting in the wavy banding pattern observed in some gels. These effects do not alter band identification or quantification and are unrelated to the western blot detection process itself.

    Reviewer 2

    Minor comments:

    We will define KChIP2.1 as suggested. We will include a diagram of the full domain structure of Kv4.3. We acknowledge that failure to do this has caused confusion for reviewer 4.

    Reviewer 3

    Minor comments:

    We will refer to ‘non-palmitoylatable mutant of Kv4.3S’ as suggested. We will carefully review the manuscript for consistency of terminology between FT293 and HEK293. Both cell lines were used in the investigation. Alongside the more detailed suggestions of reviewer 4 we will review the manuscript carefully for clarity. We will quote significant figures consistently in line with editorial guidelines.

    Reviewer 4

    *The manuscript has a flavor of LLMs… *

    We assure the reviewer that we are perfectly capable of producing unclear prose without artificial assistance. In all seriousness, we are happy to acknowledge that several sections could communicate the findings more clearly and appreciate the reviewer’s specific suggestions throughout the review. We will revise these sections to simplify wording, clarify the motivation for key experiments, strengthen transitions between sections, and improve accessibility for non-specialist readers.

    Suggested revisions to abstract & introduction…

    We will rewrite to improve readability as suggested. ‘Palmitoylation competent’ refers to the form of Kv4.3 that is recognised and palmitoylated by its palmitoylating enzyme. Kv4.3 is only ‘palmitoylation competent’ when it has been phosphorylated: we will improve the clarity of this concept in a revision.

    Suggested revisions to the results…

    We agree with the reviewer’s concerns about the dynamic range of western blotting. The detection system ensures we avoid saturation of bands. We adjust contrast for maximum clarity of presentation, but the underlying image is never saturated.

    We will include a cartoon / diagram of the full domain structure of Kv4.3 which includes the T1 domain and will improve understanding of this experiment. The T1 domain mediates tetramerisation. In its absence, Kv4.3 does not traffic to the plasma membrane.

    PEGylation to measure Kv4.3 palmitoylation stoichiometry

    We agree that acyl-PEG exchange sometimes provides useful complementary information regarding palmitoylation stoichiometry. We tried PEGylation-based approaches for Kv4.3 and were unable to obtain satisfactory resolution of the PEGylated species. In our hands, Kv4.3 is a rather messy multi-band pattern on SDS-PAGE, which makes it difficult to unambiguously resolve the mobility shifts caused by PEG addition. We suspect that both the relatively large size of the protein and its heterogeneous behaviour in a gel contribute to this challenge. We have successfully applied PEGylation methods to several smaller palmitoylated proteins (PMID 38466300). Given these technical limitations, we do not believe that PEGylation would provide a reliable orthogonal assessment of Kv4.3 palmitoylation in the present study. We have therefore continued to employ Acyl-RAC, which robustly detects relative changes in Kv4.3 palmitoylation across experimental conditions.

    Acyl-RAC experimental design?

    The acyl-RAC assay produces an ‘unfractionated lysate’ (UF) and a purified ‘palmitoylated fraction’ (P). All western blots are loaded with the same representative fraction of each. We report palmitoylation relative to expression (P/UF) and present westerns detecting the protein of interest (Kv4.3) and a known palmitoylated protein (Flot2) so the reader can judge whether a biologically meaningful fraction of the protein of interest is palmitoylated. Acyl-RAC is fundamentally an enrichment assay rather than a direct quantitative measure of occupancy. While it would be possible to estimate a percentage palmitoylation by comparison with control proteins, such calculations would, in our view, over-interpret the data. We therefore prefer to present the Acyl-RAC results as relative measures of palmitoylation normalised to protein expression rather than assign a formal stoichiometry that cannot be measured directly using this approach.

    ‘Increased palmitoylation...?’

    This refers to the fraction of Kv4.3 that is modified with palmitate. We cannot distinguish between singly and doubly palmitoylated species using Acyl-RAC. We will clarify this meaning throughout.

    Why make TMD-Del?

    We considered two possible scenarios regarding why Kv4.3 gets palmitoylated. First, palmitoylation could be promoted by membrane association of Kv4.3 through its transmembrane domains. Alternatively, palmitoylation could depend on assembly of the channel complex through the N-terminal T1 tetramerisation domain. To distinguish between these possibilities, we first examined the effect of disrupting the T1 domain. Loss of T1 abolished palmitoylation (Fig 1B), arguing against a simple requirement for membrane association. We therefore generated a minimal construct containing only the T1 domain and C-terminus with the palmitoylation sites to test this hypothesis directly. We have revised the results section to explain this rationale more clearly.

    Dephosphorylating nanobodies:

    We will improve our explanation of the rationale behind this experiment. We suggest that the impact of dephosphorylating nanobodies on Kv4.3 mobility is very clear already (lanes 1, 3, 5 of the Kv4.3 and GFP blots in Fig 2G) but would be happy to include an additional western blot.

    Section starting line 266:

    We acknowledge in the manuscript that zDHHC5 is not the only enzyme capable of palmitoylating Kv4.3 and we will emphasise that point here.

    Fig 4C: the huge enrichment of GFP-Kv4.3 achieved in the immunoprecipitation experiment makes it very hard to present lysate and IP fractions alongside each other. We will present these separately with contrast adjusted for maximum clarity.

    We will improve the justification for our focus on zDHHC5: zDHHC5 is very much more abundant in the heart than the other Kv4.3-interacting enzyme zDHHC8. We argue that the persistence of Kv4.3 palmitoylation in zDHHC5-KO cells does not exclude a physiologically important role for zDHHC5. Compensation and substrate sharing among zDHHC family members are well-recognised phenomena, often resulting in only partial reductions in substrate palmitoylation when a single enzyme is silenced / knocked out.

    … experiments on the identification of the kinases appears premature. It is unclear what the presented experiments add to the story. What in a cell is not stopped by 24hrs of staurosporin?

    We respectfully disagree. As is apparent in Figure 4F, 24hrs of staurosporine is entirely without effect on palmitoylation of Flot2. We acknowledge that we stop short of identifying the kinase responsible for priming Kv4.3 palmitoylation, but we do establish that its activity is sensitive to both serum and staurosporine. We suggest these findings merit inclusion in the manuscript.

    … acute kinase inhibition is not expected to reduce the levels of S-acylation once it has happened

    We believe the reviewer's argument assumes that the palmitoylated pool is static once formed, but our findings do not support this interpretation. The relatively rapid reduction in palmitoylation following serum withdrawal (Fig 4E) indicates that Kv4.3 palmitoylation turns over dynamically. Under these circumstances, inhibiting Kv4.3 phosphorylation is expected to reduce the replenishment of palmitoylated channels and thereby decrease steady-state palmitoylation levels, even if the turnover of palmitoylation is slower than that of phosphorylation. The fact that Kv4.3 palmitoylation reduces quickly on serum withdrawal but more slowly on kinase inhibition may indicate some influence of serum withdrawal on the palmitoylation machinery. We will clarify this point in a revised manuscript.

    the authors should monitor the phosphorylation of the acylation-deficient mutant

    This is actually already presented in the manuscript. The phosphorylation-dependent bandshift of C546/547AA TMD-del mutant is shown in Figure 2C. We will revise the manuscript to emphasise this point.

    Electrophysiological analysis of the long isoform would strength the conclusions

    We respectfully disagree. The fact that the long variant is much less palmitoylated than the short isoform makes a functional comparison of wild type and non-palmitoylated long splice variants much less valuable. We have made these recordings and find significant differences between the behaviour of wild type long and short variants that would confound the suggested experiments. A separate manuscript is in preparation.

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    Referee #4

    Evidence, reproducibility and clarity

    In this manuscript, the authors show that Kv4.3 can undergo S-acylation on Cys-546/547 in a manner that depends on phosphorylation of Ser538. They propose that this phosphorylation favors the interaction with ZDHHC5. They show that this acylation occurs differently in a long splice variant, where residues 488-498 that localize to the splice insert, inhibit acylation. They go on to showing that acylation constrains the activity of the channel.

    The findings that phosphorylation primes the channel for S-acylation, that S-acylation is reduced in the often expressed Long isoform, and that acylation control channel activity is of interest. Some less convincing aspects weaken the paper: ZDHHC5 does not appear as a major ZDHHC for K4.3., therefore one wonders why investigating the interaction mode. The work on the kinases is too preliminary. The paper would also benefit from improved writing, with the rational of each experiment is made clear, with an improve flow between sentences. The manuscript has a flavor of LLMs that tends to adds confusion rather than clarity. I might be wrong, and if so I apologies for this comment.

    The following comments should be addressed.

    Abstract:

    • To make the abstract more accessible to non-experts, the authors should mention what kChIP2.1 is.

    • The sentence lines 60-63 is unclear

    • Line 57: What does "more palmitoylated" really mean?

    • Lines 72-74: clarify that long variant undergoes limited S-acylation because its phosphorylation is reduced

    Introduction:

    • The first paragraph is difficult to follow for non-experts and might limit the breadth of the audience that will read the paper.

    • Line 134: I find the terminology "palmitoylation-competent" confusing. Do the authors mean that it gets palmitoylated? Maybe the authors implicitly refer to the fact that phosphorylation allows interaction with ZDHHC5?

    • The last sentence of the intro is quite confusing, it does not help the reader remember what the paper shows.

    Results:

    • General issue: the dynamic range of western blotting is not very large. Many of the blots have bands that go from grey to thick black bands, so beyond the dynamic range. Or is this only on the figure and not with the detection system?

    • A cartoon of Kv4.3 would be helpful.

    • What is T1? The first transmembrane domain? What is the logic of removing it? Why does this indicate that assembly precedes acylation? Here again I suggest removing the term "palmitoylation-competent" at this stage of the paper, readers cannot understand what it means.

    • Fig. 1C is too small.

    • Fig. 1E: how were the acyl RAC sample loaded? What fraction of the lysate was loaded? The fact that the long form is "more" acylated is an important conclusion of the paper. Therefore confirming this finding with alternative approaches such as PEGylation would strengthen the findings. Indeed some information on the stoichiometry would be valuable.

    • Line 162: A reference should be provided regarding the cross-talk between phosphorylation and acylation.

    • Line 188: the authors find higher WB signals by acyla-RAC. But again what does "increased palmitoylation" really mean? Do more channels acquire 2 palmitates on Cys-546/54? PEGylation would be informative.

    • Line 195: the title is quite complex/wordy.

    • What was the motivation to make the TMD-del deletion? The necessity of this entire section is unclear to me.

    • Line 210: what does this title mean?

    • Line 211: Slower band should read "slower migrating band".

    • Line 212-218: please explain the rational: you hypothesize that palmitoylation depends on phosphorylation, therefore you wish to force dephosphorylation, for this you target a general phosphatase to Kv4.3 using a nanobody system, etc...

    • What are the three complementarity-determining regions? The FLAG tag is presumably on the nanobody?

    • The authors should show Western blots of total cell lysates (in addition to the acyl-rac) blotted against Kv4.3 with LaG16 and 3W to show the impact on the slower migrating band.

    Section starting line 266

    • The beginning of this section should be reworded. Given the significant signal for Kv4.3 in ZDHHC5 KO cells (Fig. 4B), ZDHHC5 is not the major ZDHHC that modifies Kv4.3. The turbo-ID approach leads to interacting ZDHHCs but does not represent a strategy to find the ZDHHC enzyme(s) for a given substrate. So the wording should be adapted.

    • Fig. 4C is not very convincing. Lower exposures of the IP should be shown, these are outside of the dynamic range of the method.

    • Lines 282-283: S-pamitoylation does not attract proteins to membranes. Also phosphorylation only affects the binding to negatively charged membrane. This sentence is therefore incorrect in my opinion.

    • The data presented in ZDHHC5 KO cells quite convincingly shows that this is not the major acyltransferase involved. Therefore, is it justified to investigate in depth how the interaction occurs? Wouldn't it be more important to screen the ZDHHC enzyme to see it there is a major ZDHHC involved and focus on that one?

    • "several modeling scenarios" is a bit vague.

    • Why was serum starvation used? This should be explained.

    • The set of experiments on the identification of the kinases appears premature. It is unclear what the presented experiments add to the story. What in a cell is not stopped by 24hrs of staurosporin? Also, if phosphorylation is required for S-acylation, acute kinase inhibition is not expected to reduce the levels of S-acylation once it has happened.

    • To support the sequential modification of Kv4.3, the authors should monitor the phosphorylation of the acylation-deficient mutant.

    • Electrophysiological analysis of the long isoform would strength the conclusions.

    Significance

    This study adds to previous findings of the same group that cardiac channels are regulated by S-acylation and that their S-acylation is in turn controlled by signalling though the effect of kinases.

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    Referee #3

    Evidence, reproducibility and clarity

    Biochemical observations provide convincing evidence for a pathway for S-palmitoylation of Kv4.3 and its regulation as a consequence of phosphorylation. It is particularly informative that the consequences of phosphorylation are slow to develop (overnight in these experiments). The observations also show a lower S-palmitoylation state of Kv4.3L relative to Kv4.3S as a consequence of a splice insert in Kv4.3L.

    Electrophysiological observations using conventional patch techniques in HEK293 cells show that in comparison to WT a non-palmitoylatable mutant, C546/547AA, gives rise to an increased current carried by this K channel, both in response to a protocol of square wave depolarizations and in 'AP clamp' experiments using an atrial AP waveform. There were also changes in the time course of the current.

    There are limitations of the work, particularly concerning hiPSCs where the authors point out that 'palmitoylation did not measurably alter AP duration or contractility parameters in hiPSC-derived cardiomyocytes under the conditions tested'. However, the authors suggest reasons for why observations in hiPSCs differ from those expected from the work in HEK293 cells.

    The authors are aware of limitations, and a good summary of these is provided at the end of the Discussion.

    The observations are convincing and generally well presented. I recommend publication.

    Minor suggested modifications to the text are listed below to aid clarity of presentation.

    • The first point concerns the use of 'non-palmitoylatable Kv4.3S' which occurs first in the Abstract but is also used at several points later in the text. Presumably 'non-palmitoylatable' is defining rather than descriptive. In other words my understanding is that this refers to the non-palmtoylatable mutant of Kv4.3S or C546/547AA. Since Kv4.3S is palmitoylatable I suggest that 'non-palmitoylatable Kv4.3S' is replaced throughout the text to avoid confusion (e.g. in the Abstract replace 'non-palmitoylatable Kv4.3S' with 'the non-palmitoylatable mutant of Kv4.3S')

    • I also suggest consistency in the use of HEK293 or FT293. This is important for the non-specialist reader. HEK293 is used in most of the text (eg. p9 describing electrophysiology), but in Methods the cells are identified as FT293. It would be helpful also to include HEK293/FT293 in the legends to Figures 5 and 6. The legend to Figure 4E seems to be the first occurrence of FT293 while HEK293 is used earlier in the legend.

    • On p6 it is said that 'We conclude from these experiments that Kv4.3L is not less palmitoylated than Kv4.3S because of phosphorylation in the alternatively spliced region.' For ease of understanding I suggest 'phosphorylation is not the cause ...'

    • In the legends to Figures 5 & 6 the observations are quoted as four significant figures but in my opinion this is not justified on the basis of accuracy of the measurements, and is less easy to read than three. However, it seems to be common practice to use four so a change is not essential. In the legend to Figure 5F 'WT (582.81{plus minus}24.25 pA/pF) and MT (988.73{plus minus}104.58 pA/pF)' is not a justified presentation of the measurements.

    Significance

    The observations in this paper are of interest both in terms of general principles of post translational modification and the specific case of regulation of Kv4.3 channels which carry a component of the cardiac transient outward current, I(to). This functionally important current determines the shape of the cardiac action potential, particularly in its early stages, and therefore influences the magnitude and time course of contraction.

    Strengths and limitations: the approach is thorough with extensive biochemical observations, complemented by electrophysiological studies to investigate functional effects; limitations are recognised by the authors and summarised at the end of the Discussion; particular limitations are that the signalling environment within HEK293 cells is likely to differ from that in cardiac myocytes, and it is recognised that while hiPSCs have the advantage of being of human origin their precise structure and other properties differ from those of adult cardiac myocytes.

    Advance: the work increases our general understanding of post translational modification of proteins, particularly membrane proteins which control electrical activity of the heart; the work also provides important advances in our understanding of the regulation of a particular protein which is crucial for the control of electrical activity and contraction.

    Audience: the work will be of special interest to basic scientists studying cardiac physiology but also has clinical applications.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This is a well-executed study reporting a phosphorylation-dependent mechanism that preferentially primes palmitoylation of the short-form Kv4.3 potassium channel (Kv4.3S). The effect of phosphorylation on palmitoylation is splice-variant specific and is due to the absence of an inhibitory sequence element near the palmitoylated cysteines in the C-terminal disordered region of Kv4.3S. An engineered construct that links the N-terminal and T1 domains to the C-terminal cytoplasmic domains of the channel enabled detection of a phosphorylated and palmitoylated species by a slower electrophoretic mobility. This construct facilitated identification of the relevant phosphorylation site that stimulates palmitoylation. zDHHC5 was identified as a (but not the sole) palmitoyltransferase for Kv4.3S based on proximity labeling and biochemical assays. Phosphorylation facilitated the association between Kv4.3 and zDHHC5 and modeling suggests a plausible mechanism by which phosphorylation facilitates the enzyme-substrate interaction. The functional consequences of palmitoylation were studied by single cell electrophysiology assays in cell lines and action potential and contractility recordings in cardiac myocytes differentiated from inducible stem cells. Palmitoylation-deficient Kv4.3 exhibited increased current amplitude and altered gating kinetics, supporting a role for palmitoylation as a negative regulator of channel function. An effect of action potential duration or contractility parameters was not observed when palmitoylation-deficient Kv4.3 was expressed in cardiac myocytes.

    Major comments

    The conclusions of the manuscript are very well supported by the data. No concerns about reproducibility or statistical analysis. The approach is comprehensive and the manuscript presents a complete story. Interpretation of the data was thoughtful and alternative explanations offered. The section "limitations and future directions" basically covered the issues that came to mind as I was reading the paper.

    Minor comments

    I have only minor suggestions for improving accessibility of the manuscript to a broader audience. KChIP2.1 should be defined as an auxiliary protein for the channel in the abstract. The authors might also consider adding a diagram of the domain structure of full-length Kv4.3.

    Referees cross-commenting

    Agree that the reviews are fair and remarkably consistent in the issues raised.

    Significance

    Reversible palmitoylation of proteins is well characterized with respect to its effect on the palmitoylated target, i.e. a palmitoylation may change its interaction with a membrane, its trafficking, or its stability. There is a major gap in knowledge of how palmitoylation of the target is regulated by upstream signals. One mechanism that has been described is a palmitoyltransferase cascade of enzyme activation, akin to kinase cascades. This study provides a novel and convincing case of phosphorylation governing palmitoylation of a target protein, a mechanism that may be generalizable to other palmitoylation substrates.

    The study can be categorized as basic research with relevance to the fields of protein lipidation, cell signaling, and cardiovascular biology.

    My expertise is in protein lipidation, with a focus on the enzymes that catalyze protein palmitoylation. I do not have expertise to evaluate the electrophysiology experiments.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors identified a novel phosphorylation-dependent mechanism that regulates splice variant-specific S-palmitoylation of the cardiac potassium channel Kv4.3. Using complementary biochemical, proteomic, and electrophysiological approaches, they demonstrate that phosphorylation promotes Kv4.3 palmitoylation and modulates channel function. Overall, the study is of good quality and provides important mechanistic insights into the post-translational regulation of cardiac ion channels.

    Major comments:

    1. The authors used acyl-resin assisted capture (Acyl-RAC) to assess Kv4.3 palmitoylation in mouse heart and brain tissues, as well as in HEK293 cells. Although Flot2 was included as a positive control, a negative control (e.g., SAP102) is missing and should be included to validate the specificity of the Acyl-RAC assay.

    2. Statistical analysis is missing for Figure 1J. Quantification from independent biological replicates should be provided to support the conclusions.

    3. The authors state that KChIP2.1 induces the appearance of a Kv4.3 doublet in Figure 2. However, it is unclear whether KChIP2.1 was expressed in the experiments shown in Figure 2G. Moreover, doublet bands are also visible in several panels of Figure 1. Except for Figure 2B, the doublets are not clearly resolved, particularly in Figure 2G, where the authors conclude that LaG16-PP2A collapses YFP-Kv4.3S from a doublet to a single band. Extended gel-running might help support this conclusion.

    4. The TurboID analysis identified both zDHHC5 and zDHHC8 as candidate palmitoyl acyltransferases. The authors focused exclusively on zDHHC5. Is there a rationale for not investigating the potential contribution of zDHHC8?

    5. In Figure 4C, what does the y-axis represent? If the data combined multiple independent experiments, the values should be presented as normalized co-immunoprecipitation signals.

    Minor comments:

    1. The functional and mechanistic differences between KChIP2.1 and KChIP2.2 should be briefly explained in the Results section to help readers understand why only KChIP2.1 promotes Kv4.3 palmitoylation.

    2. The font size in Figures 1C and 2E is too small and should be increased to improve readability.

    3. "Kv4.3" is cut off in figure 2G.

    4. zDHHC5 is not introduced or defined.

    5. Most the western blot bands are uncommonly wavy. Is this a technical problem? Western blot method details should be provided.

    Referees cross-commenting

    Reports from the other reviewers seem fair to me. (Please note that my expertise is in electrophysiology rather than biochemistry.)

    Significance

    • The findings represent a solid biophysical advance into the post-translational regulation of cardiac ion channels which should be of considerable interest to cardiac physiologists and the ion channel community in general.

    • The review was performed by lab members experienced in neurophysiology and biophysics.

  6. Version published to 10.64898/2026.07.03.735752 on bioRxiv