
==== Front
Commun Biol
Commun Biol
Communications Biology
2399-3642
Nature Publishing Group UK London

39300259
6873
10.1038/s42003-024-06873-4
Article
Lipophilic compounds restore function to neurodevelopmental-associated KCNQ3 mutations
Edmond Michaela A. 14
Hinojo-Perez Andy 1
Efrem Mekedlawit 2
Yi-Chun Lin 2
Shams Iqra 1
Hayoz Sebastien 15
de la Cruz Alicia 16
http://orcid.org/0009-0005-7723-7860
Perez Rodriguez Marta E. 1
Diaz-Solares Maykelis 1
Dykxhoorn Derek M. 3
http://orcid.org/0000-0003-3581-754X
Luo Yun Lyna 2
http://orcid.org/0000-0003-4804-2739
Barro-Soria Rene rbarro@med.miami.edu

1
1 https://ror.org/02dgjyy92 grid.26790.3a 0000 0004 1936 8606 Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL USA
2 https://ror.org/05167c961 grid.268203.d 0000 0004 0455 5679 Department of Biotechnology and Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA USA
3 https://ror.org/02dgjyy92 grid.26790.3a 0000 0004 1936 8606 John P. Hussman Institute for Human Genomics, John T. Macdonald Foundation Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL USA
4 https://ror.org/01f5ytq51 grid.264756.4 0000 0004 4687 2082 Present Address: Texas A&M University Health Science Center, Department of Neuroscience & Experimental Therapeutics, Bryan, USA
5 https://ror.org/03m2x1q45 grid.134563.6 0000 0001 2168 186X Present Address: Department of Physiology, University of Arizona, Tucson, USA
6 https://ror.org/05ynxx418 grid.5640.7 0000 0001 2162 9922 Present Address: Linkoping University, Department of Biomedical and Clinical Sciences (BKV), Linkoping, Sweden
19 9 2024
19 9 2024
2024
7 11817 5 2024
10 9 2024
© The Author(s) 2024
2024
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A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss– and gain–of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel’s pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

Fatty acids restore the function of neurodevelopmental-associated KCNQ3 mutations that either disrupt channel function by directly affecting voltage sensor movement or by changing voltage sensor-to-pore coupling.

Subject terms

Single-molecule biophysics
Computational biophysics
Neurodevelopmental disorders
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Neuronal excitability is regulated by a variety of K+ channels, including a heterogenous population of voltage-gated K+ (Kv) channels. Kv channels are widely expressed in peripheral and central neurons, where they help to regulate resting membrane potential, action potential shape and frequency, and neurotransmitter release1. Members of the Kv7 family of channels (Kv7.2-5; also known as KCNQ channels2–4) are found in multiple neuronal types where they play a critical role in generating the sub-threshold K+ current (M-current) that regulates excitability5,6. The hyperpolarized midpoint of activation of KCNQ channels, their slow activation and deactivation kinetics during action potential initiation, and their lack of inactivation lead to sustained outward K+ currents that promote the restoration of resting membrane potential during repetitive firing. As such, these channels have the important role of providing a brake on repetitive burst firing7.

Impairment of M-currents due to mutation of KCNQ3 increases neuronal excitability and delays the development of complex neuronal networks that contribute to a variety of neurodevelopmental disorders (NDDs)8–12. The phenotypic heterogeneity of KCNQ3-associated NDDs is broad, ranging from benign familial neonatal seizures13 to more severe developmental epileptic encephalopathy with cognitive impairment14,15. Among the NDD-associated KCNQ3 variants, there is an overrepresentation of mutations in the arginine residues that serve as gating charges in the voltage sensor (S4 helix)9–11,16. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remain unknown. Therefore, a comprehensive investigation of the mechanisms by which different variants impact channel function is required to understand the etiology of these emerging KCNQ3-associated diseases.

A mechanistic understanding of KCNQ3-based channelopathies is also crucial for the development of effective therapeutic strategies. Small molecules that target neuronal KCNQ channels have been used as anticonvulsants, including pore openers such as retigabine and its derivatives7,17–19, and compounds targeting the VSD such as ICA family20–22. However, concerns over side effects23–27 have relegated these drugs to primarily research tools. Naturally occurring compounds, including medicinal plant extracts28, endocannabinoids29, and polyunsaturated fatty acids (PUFAs)30,31, have recently emerged as novel KCNQ channel regulators with differing degrees of specificity. PUFAs are particularly interesting because their amphipathic properties confer the ability to interact with lipid-soluble and -insoluble domains of proteins32,33. Indeed, ketogenic diets, which increase levels of PUFAs in both the blood34 and the brain35, have been previously used to treat children with developmental disorders30,31,36. However, whether and how PUFAs modify neuronal excitability via an effect on KCNQ channels remains unknown.

In this study, we investigated the molecular mechanisms underlying the effects of two KCNQ3 variants associated with NDD, R227Q and R236C. We found that R227Q resulted in gain-of-function (GoF) and R236C in loss-of-function (LoF). The two mutations perturbed channel gating by two distinct mechanisms: R227Q altering S4 movement and R236C altering S4-to-gate coupling. We also investigated whether PUFAs could diminish the effects of these mutations and identified two potential compounds: N-arachidonoyl amine (NAA+), which partially reversed the GOF mutation R227Q, and N-arachidonoyl taurine (NAT), which reversed the LOF mutation R236C. Molecular dynamics (MD) simulations suggested that PUFAs primarily target the VSD in its activated (S4 up) conformation and form a weaker interaction with the channel’s pore, in agreement with their ability to rescue mutations that disrupt S4 movement and S4-to-gate coupling. These results reveal how NDD-associated variants disrupt KCNQ3 channel function and suggest that lipophilic compounds could be use as efficacious, potent, and safe compounds to restore normal channel physiology.

Results

Two NDD-associated mutations in the KCNQ3 S4 segment have distinct biophysical properties

We set out to investigate the mechanisms by which variants in the voltage sensor (S4) of KCNQ3 impact channel function to cause NDDs9–11. Two missense mutations in the voltage sensor (S4) segment of the KCNQ3 channel, R227Q and R236C (Fig. 1A), were introduced into the KCNQ3 channel bearing the A315T mutation (hereafter referred to as KCNQ3), which enhances membrane insertion37,38. KCNQ3, KCNQ3-R227Q, and KCNQ3-R236C were expressed in homotetrameric (Fig. 1) and heterotetrameric (Supplementary Fig. 1) forms in Xenopus oocytes and currents recorded using two-electrode voltage clamp (TEVC) electrophysiology.Fig. 1 R227Q and R236C have distinct biophysical properties.

A Schematic of KCNQ3 channel highlighting residues analyzed in this study. B Schematic of recording setup for TEVC. Images in (A) and (B) were generated using UCSF ChimeraX, version 1.1 (2020-10-07) and CorelDraw Graphics Suite 2021 software, respectively. Representative current traces from KCNQ3-A315T-R227Q (R227Q) (C), KCNQ3-A315T (wt) (D), and KCNQ3-A315T-R236C (R236C) (E) channels for the indicated voltage protocols. F Extrapolated tail conductance from panels C, D, and E were normalized and plotted against test voltages to create G(V) curves (R227Q closed circles; wt open diamonds; R236C closed squares). Lines represent the fitted theoretical voltage dependencies (Eqs. 1 and 2). G Summary data for G(V) midpoints using Boltzmann fits from panel F. H Representative current time courses of R227Q, wt, and R236C channels in response to the indicated protocol. The dashed line represents 50% of maximum current at the end of the depolarizing pulse. I Time course of current activations quantified as the time to reach half maximum current at the end of the depolarizing pulse in (H, dashed line). J Summary of ΔΔG data (Eq. 5) for R227Q and R236C channels. Midpoints of voltage activation and ΔΔG values for each channel are shown in Supplementary Table 1 and Supplementary Data 1. Data presented as mean ± SEM, n = 5–11. Statistical significance determined using one-way ANOVA and Bonferroni’s post hoc test, p < 0.05.

In comparison to homomeric KCNQ3 channels, we found that homomeric R227Q channels displayed a hyperpolarizing (leftward) shift in their steady-state conductance-voltage G(V) curve and an accelerated time course of current activation (Fig. 1C, F–I, Supplementary Table 1, and Supplementary Data 1), in agreement with previous reports9. Indeed, homomeric R227Q channels remained open throughout the physiological voltage range (from –100 to 0 mV, Fig. 1F). In contrast, homomeric R236C channels displayed a depolarizing (rightward) shift in their G(V) curve and a slower time course of current activation (Fig. 1E–I, Supplementary Table 1, and Supplementary Data 1). To better compare the functional effect of G(V) shifts on these mutants, we corrected for the differences in slopes and calculated the change in Gibbs free energy (∆∆G,) to estimate the energy required to open each channel39,40. Compared to homomeric KCNQ3 channels, R227Q decreased the energy required for channel opening whereas R236C increased it (Fig. 1J, Supplementary Table 1, and Supplementary Data 1), consistent with GoF and LoF, respectively.

Co-expression of homomeric R227Q or R236C mutations with wt KCNQ2 and wt KCNQ3 subunits –to mimic heteromeric channels in heterozygous patient cases– revealed a gene-dose effect for these mutations, such that the biophysical phenotype was more severe with a greater number of mutated subunits per channel (Supplementary Fig. 1 and Supplementary Table 3). To avoid any confounding effects of subunit composition, we proceeded to investigate the mechanisms underlying GoF and LoF in these two mutants using homomeric KCNQ3 channels.

R227Q and R236C affect channel function through different mechanisms

Given the effect of R227Q and R236C on voltage gating, we measured S4 movement and channel opening simultaneously using voltage clamp fluorometry (VCF). Wt and variant-bearing KCNQ3 channels were fluorescently labelled by attaching Alexa-488-maleimide to the Q218C site in the S3-S4 loop (hereafter referred to as KCNQ3L) (Fig. 2A). This manipulation has previously been shown to faithfully report S4 movement41,42. The KCNQ3L construct produced a voltage-dependent fluorescence change, F(V), that was correlated with the G(V) curve (Supplementary Fig. 2). Interestingly, KCNQL-R227Q channels also exhibited G(V) and F(V) signals that closely followed each other but were left shifted compared to KCNQ3L (Fig. 2B, C, Supplementary Table 1, and Supplementary Data 1). In contrast, the G(V) and F(V) curves of KCNQL-R236C channels were right-shifted compared to KCNQ3L, although the F(V) curve showed an intermediate shift that did not overlap with the G(V) curve (Fig. 2D, E, Supplementary Table 1, and Supplementary Data 1). The changes in Gibbs free energy associated with S4 activation and channel opening in R227Q- and R236C-containing channels were consistent with their GoF and LoF phenotypes, respectively (Supplementary Table 1 and Supplementary Data 1). Together, these results suggest that R227Q and R236C shift G(V) curves by interfering with different gating transitions. The alignment of F(V) and G(V) curves at hyperpolarized voltages for the R227Q variant, together with the decrease in free energy required to activate S4, suggest that R227Q directly affects S4 movement. In contrast, the distinct F(V) and G(V) curves for the R236C variant, and the increased energy required to both move S4 and open the channel, is indicative of an alteration in S4-to-gate coupling.Fig. 2 R227Q and R236C affect channel function by different mechanisms.

A Schematic representing VCF technique. A cysteine introduced at position 218 (close to S4) is labeled with a fluorophore tethered to Alexa-488–5 maleimide. Upon voltage changes, labeled-S4s move and the environment around the tethered fluorophore changes, altering fluorescence intensity. Current and fluorescence are recorded simultaneously using the setup shown on the right. Location of the two NDD–associated mutations (R227Q and R2236C) are shown on the left. Cartoons in A were generated using CorelDraw Graphics Suite 2021 software. B Representative current (black) and fluorescence (cyan) traces from Alexa-488–labeled KCNQ3-A315T-R227Q (R227Q) channels for the indicated voltage protocol. C Normalized G(V) (black circles and black solid line from a Boltzmann fit) and F(V) (cyan circles and cyan solid line from a Boltzmann fit) curves from labeled R227Q. D Representative current (black) and fluorescence (blue) traces from Alexa-488–labeled KCNQ3-A315T-R236C (R236C) channels for the indicated voltage protocol. E Normalized G(V) (black squares and black solid line from a Boltzmann fit) and F(V) (blue squares and blue solid line from a Boltzmann fit) curves from labeled R236C. Dashed lines represent labeled pseudo-wt KCNQ3-A315T-Q218C G(V) (gray) and F(V) (black) curves for comparison (raw data shown in Supplementary Table 1, Supplementary Fig. 2, and Supplementary Data 1). Midpoints of voltage activation are shown in Supplementary Table 1 and Supplementary Data 1. Data represent mean ± SEM. n = 5–13.

PUFAs can activate or inhibit KCNQ3 channel function

Because the R227Q and R236C mutations alter S4 movement and gate opening via different mechanisms, we hypothesized that different strategies would be required to restore channel functionality. We, therefore, screened a diverse panel of naturally occurring PUFAs and PUFA analogs for their ability to modulate G(V) relationships, current amplitudes (Gmax), and kinetics of activation and deactivation in KCNQ3 channels expressed in Xenopus oocytes (Fig. 3 and Supplementary Fig. 3).Fig. 3 PUFAs can activate or inhibit KCNQ3 channel function.

Representative current traces from KCNQ3-A315T channels in the absence (before) or presence (after) of 25 μM NAA+ (A) or NAT (C) for the indicated voltage protocol. B, D Steady-state G(V) curves (solid lines from a Boltzmann fit) obtained from recordings in panels A and C normalized to peak conductance before PUFA application (open symbols). G(V) relationships (solid lines from a Boltzmann fit) in the presence of PUFAs are shown as closed symbols and dashed lines represent their respective normalized G(V). Summary data for shifts in half-activation voltage (ΔV1/2) for each G(V) (E), increase in Gmax (ΔGmax) (F), ΔΔG (G), and relative change in potassium current at –60 mV (ΔI–60) (H) induced by 25 μM PUFAs on KCNQ3 channels. Dashed lines represent DMSO-induced changes for comparison. Representative time courses of current activation (I) and deactivation (J) in the absence (black) and presence of NAA+ (red) and NAT (blue) in KCNQ3-A315T channels in response to the indicated voltage protocol. Dashed lines represent 50% maximum current level at the end of the depolarizing pulse. K Time courses of current activation (closed symbols) and deactivation (open symbols) in the absence (black) and presence of NAA+ (red) and NAT (blue) quantified as time to reach half maximum current level at the end of the depolarizing pulse in (I and J, dashed lines). Values for V1/2, ΔV1/2, Gmax (Eq. 2), ΔΔG (Eq. 5), ΔI-60 (Eq. 3), and time to I50% given in Supplementary Table 2 and Supplementary Data 1. Data represent mean ± SEM; n = 6–9. Statistical significance determined using Student’s T-test to compare the PUFA–induced change in ΔV1/2, ΔGmax, ΔΔG, ΔI–60, and time to I50% relative to the mock application of a solution containing only vehicle (DMSO); only significant differences are shown. One-way ANOVA and Bonferroni’s post hoc test, containing the other 5 screened PUFAs and PUFA analogs in Supplementary Fig. 3, also gave p < 0.05 for NAA+ and NAT (see Supplementary Fig. 3).

Among the PUFAs tested, two PUFA analogs, N-arachidonoyl amine (NAA+) and N-arachidonoyl taurine (NAT–), had more substantive effects on KCNQ3 channels. 50 μM NAA+ right shifted the G(V) relationship by ~5 mV, reduced Gmax by 27% and increased the Gibbs free energy (∆∆G) required for channel opening by ~+4.2 kJ/mol (Fig. 3A, B, E–G, Supplementary Table 2, and Supplementary Data 1). We also quantified the amount of potassium current at –60 mV (I–60), near the action potential threshold for most neurons, and found that NAA+ reduced I–60 by 40% (Fig. 3H, Supplementary Table 2, and Supplementary Data 1). NAA+ also slowed the time course of current activation and accelerated the time course of current deactivation (Fig. 3I–K Supplementary Table 2, and Supplementary Data 1). In contrast, 25 μM NAT– activated KCNQ3 channels by left shifting the G(V) relationship by ~19 mV, increasing Gmax, decreasing the energy required for channel opening by ~7 kJ/mol, and increasing I–60 by 12-fold (Fig. 3C–K, Supplementary Table 2, and Supplementary Data 1). In addition, NAT– slightly accelerated the time course of current activation and slowed the time course of current deactivation (Fig. 3I–K, Supplementary Table 2, and Supplementary Data 1). Note that the PUFA effect reached steady-state levels within a few minutes (Supplementary Fig. 4).

Thus, although both NAA+ and NAT– modulate KCNQ3 channels, they act via mechanisms that inhibit and activate channel function, respectively. Notably, the additional PUFAs tested –docosahexaenoic acid (DHA), linoleic acid (LA), arachidonic acid (AA), and arachidonoyl ethanolamine (O-AEA)– had minimal effect on KCNQ3 channel functionality (Supplementary Fig. 3 and Supplementary Table 3).

PUFAs primarily target the KCNQ3 voltage sensor

Given its activating effect, we wondered whether NAT– might activate KCNQ3 channels by interacting with the proposed binding site for the KCNQ channel openers retigabine43 and ICA-06967344. These compounds bind to W265 (in the S5 helix), and P211 and L198 residues (in the voltage-sensing domain, Supplementary Fig. 5A). We therefore tested the effect of NAT– on KCNQ3-W265L, KCNQ3-P211N, and KCNQ3-L198F channels, and observed similar activation to that seen in wt KCNQ3 channels (Supplementary Fig. 5A and Supplementary Table 3). These data suggest that the activating effects of NAT– are not dependent on W265, P211, or L198 and therefore that NAT– activates KCNQ3 channels via a distinct mechanism.

To identify the sites at which NAT– binds to KCNQ3 channels, we conducted all-atom MD simulations of two KCNQ3 homology models in the active (S4 up) and resting (S4 down) conformation in a solvated POPC bilayer. We incorporated 20 NAT– molecules in the upper leaflet and 23 PIP2 in the lower leaflet, each corresponding to ~2.5% of the bilayer composition. While our bilayer model deviates from a native condition in which >100 lipid types are present in the plasma membrane, this type of simple binary or tertiary atomistic membrane model has been successfully used to identify putative lipid binding sites by us and others for prioritizing mutagenesis experiments45–47. Pairwise nonbonded interactions between NAT– and protein residues were analyzed for three replicas of 600 ns production runs (for the active ‘S4 up’ state) and four replicas of 1.2 μs production runs (for the resting ‘S4 down’ state) (Fig. 4A, Supplementary Fig. 6, and Supplementary Data 1). Of all 262 residues, the outermost gating charge in S4, R227, clearly had the strongest electrostatic interaction with the anionic headgroup of NAT– in all active state simulations. A secondary binding region was formed from K146 in the VSD and K284 in the pore domain (Fig. 4A, B and Supplementary Data 1). The accumulated sampling regions of NAT– around KCNQ3 are visualized in Fig. 4C, with the highest NAT– headgroup and tail density shown in 3D volume map. R227, K146, and K284 were also the strongest binding residues in the resting state simulations, but their interactions with NAT– were weaker, even though the simulations were twice as long (Supplementary Fig. 6B).Fig. 4 PUFAs primarily target the KCNQ3 voltage sensor.

A MD simulation data showing pairwise electrostatic interaction between KCNQ3 active (S4 up) state and NAT. Data points represent the electrostatic interaction from one of 200 snapshots over 2 μs. Only favorable interactions < −2 kcal/mol are shown for clarity. B Top three binding residues illustrated on one of the KCNQ3 subunits. C Volumetric map of NAT density (headgroup in yellow and tail in cyan). In the view from above (left), positions of NAT within 10 A¨ of KCNQ3 from 800 snapshots over 2 μs were overlapped. Representative current traces from KCNQ3-A315T-R227Q (D), KCNQ3-A315T-K284Q (F), KCNQ3-A315T-K146Q (H), and KCNQ3-A315T-R227Q-K284Q-K146Q (J) channels in the absence (before) or presence (after) of 25 μM NAT for the indicated voltage protocol. (E, G, I, and K) Steady-state G(V) relationships (solid lines from a Boltzmann fit) obtained from recordings in panels D, F, H, and J normalized to peak conductance before (open symbols) and after (closed symbols) NAT application, respectively. Dashed lines represent respective normalized G(V) relationships. Summary data for ΔV1/2 (L); ΔGmax (M); and ΔΔG (N) induced by 25 μM NAT for the indicated KCNQ3 mutation. Gray dashed boxes represent NAT–induced change of ‘wt’ KCNQ3-A315T in L ΔV1/2, M ΔGmax, and N ΔΔG for comparison. Values are given in Supplementary Table 2 and Supplementary Data 1. Data represent mean ± SEM; n = 5–8. Statistical significance determined using one-way ANOVA and Bonferroni’s post hoc test to compare the NAT–induced change in ΔV1/2, ΔGmax, and ΔΔG. p < 0.05; only significant differences are shown.

We tested the importance of R227 for NAT– binding using the charge-neutralizing mutation R227Q (Fig. 4B). In contrast to its effect on wt KCNQ3 channels, 25 μM NAT– was unable to shift the KCNQ3-R227Q G(V) curve or change the energy required to open these channels (Fig. 4D, E, L, M, Supplementary Table 2, and Supplementary Data 1). This suggests that S4, particularly R227, is important for mediating the effect of NAT– on voltage dependence and, therefore, that this domain may form the primary PUFA binding site. However, because NAT– remained able to increase Gmax in KCNQ3-R227Q channels (Fig. 4E, M, Supplementary Table 2, and Supplementary Data 1), we explored whether K146 and K284 may form a second binding site for this PUFA. We first made, one at a time, the neutralizing mutations K146 and K284Q (Fig. 4B). Compared to wt, the K284Q and K146Q mutations significantly reduced the ability of NAT– to left shift the G(V) curve, lowered the energy required to open the channels, and reduced the ability of NAT– to increase Gmax by ~45% and ~50%, respectively (Fig. 4F–I, L–N, Supplementary Table 2, and Supplementary Data 1). Remarkably, compared to wt channels, neutralizing all three residues (R227Q/K146Q/K284Q) not only abolished the G(V) shift cause by NAT–, but also almost completely reduced the ability of NAT– to increase Gmax (Fig. 4J–M, Supplementary Table 2, and Supplementary Data 1), indicating that both residues, K284 and K146, are critical for the increase in current amplitude (Gmax) induced by NAT–.

Taken together, these data suggest that NAT– activates KCNQ3 channels involving two distinct regions of the channel. R227 on the N-terminal region of S4, and to a lesser extent K146 and K284, are involved in the shift in voltage dependence (leftward G(V) shift) mediated by NAT–. In contrast, the extracellular regions of S1 and S5 (K146 and K284 respectively), but not S4, are responsible for the increase in current amplitude (Gmax) induced by NAT–.

PUFAs rescue function of NDD-associated KCNQ3 mutations

Because NAA+ and NAT– were the most potent inhibitor and activator, respectively, in our screen on KCNQ3 channels, we tested whether they could rescue the functionality of R227Q- and R236C-containing channels. 25 μM NAA+ modestly restored the G(V) curve of KCNQ3-R227Q channels to more positive voltages (Fig. 5A, B, E, Supplementary Table 2, and Supplementary Data 1). NAA+ also reduced both Gmax (by ~15%) and the amount of potassium current at negative voltages (Fig. 5A, B, F, G, Supplementary Table 2, and Supplementary Data 1). Increasing NAA+ to 50 μM resulted in a larger reduction of Gmax (by ~30%) but had no additional effect on the G(V) curve in R227Q channels (Fig. 5B, Supplementary Table 2, and Supplementary Data 1). Consistent with its inhibitory effect, NAA+ increased the energy required to open R227Q channels (Fig. 5H, Supplementary Table 2, and Supplementary Data 1), slowed the time course of current activation, and accelerated the time course of current deactivation (Supplementary Fig. 7A, B, E, and Supplementary Table 2).Fig. 5 PUFAs rescue NDD-associated mutations.

Representative current traces from R227Q (A) and R236C (C) channels in the absence (before) or presence (after) of 25 μM NAA+ (A, red) and NAT (C, blue) for the indicated voltage protocol. Steady-state G(V) relationships (solid lines from a Boltzmann fit) obtained from recordings in panels A and C normalized to peak conductance before (open symbols) and in the presence (closed symbols) of 25 μM and 50 μM NAA+ (B) and 25 μM NAT (D). Dotted gray lines represent G(V) curves for ‘wt’ KCNQ3-A315T channels and dashed lines represent respective normalized G(V) relationships after application of the indicated PUFA. Summary data for ΔV1/2 (E); ΔGmax (F); ΔI–60 (G); and ΔΔG (H) induced by 25 μM NAA+ (red) in R227Q channels and 25 μM NAT (blue) in R236C channels. Dashed lines represent DMSO-induced changes for comparison. Data represent mean ± SEM, n = 5–9. Values are given in Supplementary Table 2 and Supplementary Data 1. Statistical significance was determined using Student’s T-test to compare the PUFA–induced change in ΔV1/2, ΔGmax, ΔΔG, ΔI–60, and time to I50% relative to mock application of a solution containing only vehicle (DMSO) for the indicated mutation. p < 0.05; only significant differences are shown.

In contrast, treatment of R236C-bearing channels with 25 μM NAT– restored the G(V) curve to more negative voltages (Fig. 5C–E, Supplementary Table 2, and Supplementary Data 1). NAT– also increased Gmax by ~30% and potentiated potassium currents by more than 7-fold at physiologically relevant voltages (Fig. 5F, G, Supplementary Table 2, and Supplementary Data 1). Moreover, NAT– decreased the energy required to open R236C channels by ~4 kJ/mol (Fig. 5H, Supplementary Table 2, and Supplementary Data 1), accelerated the time course of current activation, and slowed the time course of current deactivation (Supplementary Fig. 7C–E, and Supplementary Table 2). Together, these data indicate that NAA+ can partly rescue the GoF phenotype induced by R227Q and, importantly, that NAT– can rescue the LoF phenotype induced by R236C so that this variant more closely resembles the properties of wt KCNQ3 channels.

PUFAs act by modifying S4 movement and channel gating

To determine whether PUFAs rescue the biophysical properties of R227Q and R236C variants due to an effect on S4 movement, gate, or S4-to-gate coupling, we used VCF to measure S4 movement and channel gating simultaneously (Fig. 6). Similar to its effect on unlabeled R227Q, 25 μM NAA+ reduced the current carried by Alexa-488-labeled KCNQ3L-R227Q channels (Fig. 6A, B, Supplementary Table 2, and Supplementary Data 1) and shifted G(V) and F(V) curves to more positive potentials (Fig. 6B, C, Supplementary Table 2, and Supplementary Data 1). NAA+ also moderately slowed the time course of current activation and did not alter the time course of current deactivation (Supplementary Fig. 8A, B and Supplementary Table 2). Furthermore, VCF revealed that NAA+ did not alter the time course of fluorescence signal (S4) activation, but accelerated the time course of fluorescence signal deactivation, compared to untreated KCNQ3L-R227Q channels (Fig. 6D, E, Supplementary Table 2, and Supplementary Data 1). Additionally, NAA+ increased the energy required to open labeled KCNQ3L-R227Q channels, but moderately increased the energy required for S4 movement (Supplementary Table 2 and Supplementary Data 1) and slightly decreased the amplitude of fluorescence in labeled KCNQ3L-R227Q channels (Fig. 6A). The mechanism by which NAA+ quenches fluorescence is unclear, but it is plausible that it induces a rearrangement of the channel that positions a nearby quenching residue, like tryptophan, closer to the fluorophore. Alternatively, NAA+ may directly reduce the fluorescence signal, or may form tiny lipid vesicles that induce a transition from aqueous to lipid medium thereby altering the microenvironment around the tethered fluorophore and, in so doing, reduce the fluorescence signal as previously suggested48.Fig. 6 PUFAs act by modifying S4 movement and channel gating.

Representative current (top) and fluorescence (bottom) traces from Alexa-488–labeled KCNQ3L-R227Q (R227Q) (A) and KCNQ3L-R236C (R236C) (F) channels in the absence (before) or presence (after) of 25 μM NAA+ (red) and NAT (blue) for the indicated voltage protocol. Steady-state G(V) relationships (black circles and black solid line from a Boltzmann fit) and F(V) relationships obtained from recordings in panels A (red circles and red solid line from a Boltzmann fit) and F (blue squares and blue solid line from a Boltzmann fit), normalized to peak conductance and fluorescence before PUFA application (open symbols). Dotted and dashed-dotted lines represent G(V) and F(V) curves for untreated R227Q (B) and R236C (G) channels, respectively. Dashed lines represent respective normalized G(V) relationships after the application of the indicated PUFA. C, H Summary data for ΔV1/2 (black) and ΔF1/2 (red and blue) obtained from recordings in panels B and G. Representative time courses of fluorescence activation (top) and deactivation (bottom) in the absence (black) and presence of NAA+ (red) or NAT (blue) from KCNQ3L-R227Q (R227Q) (D) and KCNQ3L-R236C (R236C) (I) channels in response to the indicated voltage protocol. Dashed lines represent 50% of maximum fluorescence at the end of the pulse. E Time courses of fluorescence activation and deactivation in the absence (open black symbols) and presence (closed symbols) of NAA+ (red) or NAT (blue) are quantified as time to reach half maximum fluorescence at the end of the pulse (dashed lines). Values are given in Supplementary Data 1. Data represent mean ± SEM, n = 3–5. Statistical significance was determined using pair-sample Student’s t-test. p < 0.05; only significant differences are shown.

In contrast, NAT– led to a leftward shift of both the G(V) and F(V) relationships (Fig. 6F–H, Supplementary Table 2, and Supplementary Data 1) and accelerated the time course of current activation (without affecting deactivation) in KCNQ3L-R236C channels (Supplementary Fig. 8A and Supplementary Table 2). Moreover, NAT– accelerated the time course of fluorescence activation (S4 movement), but did not affect the time course of fluorescence deactivation in KCNQ3L-R236C channels (Fig. 6I, E, Supplementary Table 2, and Supplementary Data 1). Consistent with its positive effect on activity, NAT– decreased the energy required to move S4 in KCNQ3L-R236C (Supplementary Table 2 and Supplementary Data 1).

Taken together, our VCF data suggests that NAT– effectively activates KCNQ3-R236C channels by altering S4 movement and channel gating. NAA+ had a more moderate effect on KCNQ3-R227Q channels, likely because the mutation itself removed the positive charge at position 227 which is important for NAA+ to shift the G(V) curve.

Discussion

Pathogenic variants in KCNQ3 (M-channel) subunits have been recently associated with NDDs, including devastating developmental and epileptic-encephalopathy10,11. However, the impact these KCNQ3-channelopathies have on M-channel function and NDD pathogenesis remains poorly defined, and existing treatments are often ineffective or have serious side effects limiting their utility26,49. We have investigated the functional consequences of two mutations in the voltage sensor of KCNQ3 that are linked to NDDs and autism. We found that R227Q shifts the voltage dependence of channel opening towards negative voltages by affecting S4 movement while R236C shifts the voltage dependence of channel opening towards positive voltages by altering S4-to-gate coupling. Additionally, we found that two PUFAs, NAA+ and NAT–, can partially rescue the R227Q and R236C phenotypes, respectively. The mechanisms by which PUFAs act on KCNQ3 and KCNQ3-bearing pathogenic variants were examined using all-atom MD simulations and electrophysiology. Our data suggest that the outermost positive gating charge in the voltage sensor (R227) and positively charged residues in the extracellular regions of S1 and S5 (K146, K284), but not the retigabine- and ICA-binding pockets, are critical for mediating PUFAs effects. The R227Q –and to a lesser extent K284Q and K146Q– mutants abolished the effect of NAT– on voltage dependence, supporting the hypothesis of an electrostatic interaction between the negative charge of the PUFA head group and S4 in KCNQ3.

The molecular bases of NDDs remain largely unknown. Recent studies have provided evidence linking voltage-gated ion channels, including KCNQ channels, with the pathophysiological features of NDDs50,51. The KCNQ3-R227Q mutation has been reported to cause sleep-activated near-continuous multifocal spikes9, as well as ASD or autistic features10,16. Our VCF data showed that the R227Q mutation shifts the F(V) curve (representing S4 movement) to similar negative voltages as the G(V) curve (representing gate movement). In addition, the time course of S4 movement and channel opening in R227Q are closely correlated, similar to wt KCNQ3 channels. These results indicate that KCNQ3-R227Q channels exhibit a GoF phenotype, and that channel function is altered by disturbing S4 activation and not by affecting the relationship between S4 movement and channel gating.

We recently showed that neutralization of the first gating charge in the related KCNQ2 channel (R198Q) resulted in a similar leftward shift of the G(V) curve and an overlap of this curve with the F(V) relationship52. This close correlation between G(V) and F(V) curves was reflected in the similar time courses of ionic currents and fluorescence signals52, as we observed for the KCNQ3-R227Q variant in the current study. Similar to KCNQ3-R227Q channels, previous studies using systematic mutagenesis and molecular modeling of S4 in other KCNQ family members demonstrated that mutations of the first and second gating charge of S4 caused a leftward shift in the voltage G(V) curve53–55. The proposed mechanism underlying the gain of voltage sensitivity in these mutations was the destabilization of the resting state configuration of S4. Furthermore, our earlier work showed that neutralization of the second arginine within S4, which has also been linked to NDDs9, disrupted KCNQ3 channel function by shifting the voltage dependence of S4 movement to extreme negative potentials driving the open/closed transition of the gate to very negative potentials41. Therefore, basic residues in the first and second gating charges of S4 are crucial for stabilizing the resting state of the voltage sensor and keeping the channel closed; a mechanism that seems to be conserved across KCNQ channels52,55–57.

In contrast to mutations in the N-terminal part of S4, mutations of charged residues in the C-terminal region of S4 (third, fourth, and fifth gating charges) have been reported to reduce KCNQ channel activity (LoF), primarily by shifting the G(V) curve toward depolarized voltages and either slowing the kinetics of activation or accelerating the kinetics of deactivation16,58. Motivated by these findings, we used VCF to examine how the R236C mutation in KCNQ3 affected channel activity. In contrast to R227Q, the R236C variant caused a dissociation of S4 movement and channel opening, observed as a more hyperpolarized F(V) curve than G(V) curve, suggesting that R236C altered S4-to-gate coupling. Previously52, a similar outcome was seen for the epilepsy-associated R214W mutation in KCNQ2 channels, suggesting similarities between KCNQ2 and KCNQ3 gating mechanisms. However, KCNQ2-R214 is in the loop connecting S4 and the S4-S5 linker and is thought to coordinate the binding of PIP2 – a phospholipid that regulates channel gating52 –whereas KCNQ3-R236 is in the S4 segment– two helical turns above the homologous R214 residue – and is therefore unlikely to form part of the PIP2 binding pocket.

Our study also identified PUFAs that modulate KCNQ3 channel activity restoring functionality in NDD-associated KCNQ3 channel variants. The two most effective PUFAs –NAT– and NAA+– modified voltage sensitivity, maximum current amplitude, and both activation and deactivation kinetics. Previous studies in shaker59,60 and cardiac KCNQ161 channels showed that charged PUFA analogs form electrostatic interactions with positively charged arginines in the N-terminal region of S4 to activate these channels. Our finding that the R227Q mutant reduced KCNQ3 channel sensitivity to NAT– and NAA+ suggests that the negatively charged taurine group of NAT– and the positively charged amino group of NAA+ may attract and repel the guanidinium group of R227 to stabilize and destabilize the activated state of S4, respectively. This would explain why NAT– functions as a channel activator and NAA+ as an inhibitor, and furthermore, why NAA+ had a minimal effect on S4 movement and voltage dependence but more effectively accelerated current deactivation and reduced maximum current amplitude. NAA+ caused only a small change in the time course of S4 movement, likely due to the absence of electrostatic repulsion between R227Q (in the S4) and NAA+.

VCF showed that NAT– shifted the properties of R236C mutant channels towards those of wt KCNQ3 channels, including the voltage dependence and time course of S4 movement and channel opening. Since R236C changes the coupling of voltage sensor movement and gate opening, it is tempting to speculate that NAT– may impact both the S4 and the gate, as previously suggested for KCNQ162. In KCNQ3, we found that NAT– interacts with R227 (N-terminal region of S4), a position distinct from the retigabine and ICA binding sites, resembling the mechanism of action proposed for endocannabinoids in KCNQ2/3 channels29. It is possible that the negative taurine moiety of NAT– may be attracted to the charged residues of S4, particularly R227, thereby stabilizing the activated state of S4 via electrostatic mechanisms. This would indirectly exert a restoration of the voltage dependence of KCNQ3 channel opening and restore the function of the KCNQ3-R236C mutant.

In addition of NAT’s interaction with S4 of KCNQ3, MD simulations also revealed a secondary interaction with K146 and K284, located in the extracellular loops of S1 and S5, between the VSD, lipids, and extracellular side of the pore. These data are consistent with our findings that K146Q and K284Q almost completely abolish the effect of NAT– on Gmax and voltage dependence, indicating that NAT– could also form electrostatic interactions with positively charged residues on the pore domain. Previous MD simulations of PUFAs have identified electrostatic interactions with the pore and VSD domains of KCNQ163. Interestingly, our MD simulations show that NAT– binds more strongly to S4 in the activated (up) state than the resting (down) state, likely due to a greater exposure of the R227 binding site to the membrane surface in the upstate. Such state-dependent binding suggests that NAT– stabilizes the activated conformation of S4, consistent with the slower deactivation kinetics of KCNQ3 in the presence of NAT–. From a therapeutic standpoint, PUFA’s interaction with ion channels is relatively nonspecific and, thus not ideal for clinical usage at this stage. Our long-term goal is to use the PUFA binding site that we identified here to design PUFA derivatives with higher specificity and affinity.

Lipophilic compounds like PUFAs, cholesterol, and flavonoids have been reported to alter membrane fluidity and, hence, may indirectly modify the biophysical properties of membrane proteins64–66. For instance, while flavonoids can directly act on ion channels67,68, flavonoid derivatives could also indirectly affect their gating properties through mechanisms involving a decrease in membrane fluidity by augmenting the packing order of membrane lipids69. Although PUFAs could have some effect on the biophysical properties of the membrane and, indirectly, integrated membrane proteins, our results suggest that NAA+ and NAT- act directly on the KCNQ3 channel. For example, NAA+ and NAT- each modified distinct biophysical parameters of the KCNQ3 channel in a manner dependent on the site of the different mutation, as exemplified by the distinctive effect of NAT– on the mutants R227Q (abolishing the V1/2 shift) and K284Q/K146Q (abolishing the Gmax increase). This suggests that the PUFAs act directly on the channel rather than indirectly through effects on the plasma membrane.

Our study reveals that the molecular mechanisms underlying KCNQ channelopathies could be heterogeneous, and that predicting their functional consequences could be challenging. For instance, it is often assumed that mutations affecting VSDs change the voltage-dependence of opening and result in a concomitant alteration of opening probability, whereas mutations in the pore region hamper permeability. However, our data shows that, although both R227 and R236 are located in S4 and are linked to similar NDD phenotypes11, the mechanisms by which mutations of these residues perturb channel gating are different. An understanding how mutations affect different regions of a channel is critical for the design of appropriate therapeutic strategies to treat these disabling conditions while minimizing potential side effects.

Most NDD-associated missense variants in the KCNQ3 channel alter arginine residues in the VSD9,11. Although the majority of these alterations that have been characterized lead to GoF9, likely by direct disturbance of S4, some variants (including R236C) lead to LoF due to dissociation of S4 movement and gate opening, resulting in neuronal hyperexcitability. The antagonistic biophysical properties of NAA+ and NAT– can compensate for the enhanced and reduced activity of NDD variant-bearing channels. The divergent molecular properties of these arachidonic acid derivatives could therefore be used as scaffold molecules to design more potent, and patient-specific, drugs to treat neurological disorders associated with KCNQ channel defects.

Methods

Chemicals

(ω-3) 4,7,10,13,16,19-all-cis-docosahexaenoic acid (DHA); (ω-6) 9, 12-cis- linoleic acid (LA); (ω-6) 5,8,11,14-all-cis- arachidonic acid (AA); 4,7,10,13-cis-arachidonoyl ethanolamine (O-AEA); 9, 12-cis- linoleoyl-glycine (Lin-gly); and 5,8,11,14-all-cis-N-arachidonoyl taurine (NAT) were purchased from Cayman Chemicals Inc (Ann Arbor, MI, USA). 5,8,11,14-cis- N-arachidonoyl amine (NAA+) was synthesized at Linkoping University, Sweden. Alexa Fluor 488 C5- maleimide was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). PUFAs were kept at −20 °C as 50 mM stock solutions in DMSO (NAT) or ethanol (NAA+) and diluted shortly before experiments as previously described59. Control and test solutions were added to the recording chamber using the Rainin Dynamax peristaltic pump-driven (model RP-1) perfusion system.

Molecular biology

The full-length human KCNQ3 construct (NCBI Reference Sequence: NP_004510.1; GI:4758630) was synthesized (GenScript USA, Piscataway, NJ) and ligated between the BamHI and XbaI sites in the multiple cloning sites of into the pGEM-HE vector. This vector had been previously modified to contain a T7 promoter and 3’ and 5’ untranslated regions from the Xenopus β-globin gene41. The Kozak consensus sequence (GCCACC) and the AgeI restriction site (ACCGGT) were added before the start codon (ATG) of the KCNQ3 gene. Point mutations were made in the KCNQ3 gene using the Quikchange XL site-directed Mutagenesis kit (Agilent) according to the manufacturer’s protocol. The correct incorporation of the specific variant was assessed by Sanger sequencing (sequencing by Genewiz LLC, South Plainfield, NJ). The RNA was synthesized in vitro using the mMessage mMachine T7 RNA Transcription Kit (ThermoFisher Scientific) from the linearized cDNA. mRNA (40–50 nL) was injected into defolliculated Xenopus leavis oocytes (purchased from Ecocyte) using a Nanoject II nanoinjector (Drummond Scientific) and electrophysiological experiments were performed 2 to 5 days after injection.

TEVC and VCF experiments

were performed 2–5 days after injection as previously described41,52. ND96 solution contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH = 7.5). Currents were recorded using a (TEVC) OC-725C oocyte clamp (Warner Instruments) or (VCF) Axoclamp900A amplifier (Molecular Devices), low-pass filtered at 1 kHz and sampled at 5 kHz. Microelectrodes had resistances 0.3–0.5 MΩ when filled with 3 M KCl. Oocytes were labeled for 30 min with 100 μM Alexa-488 maleimide in ND98 at 4 °C, in the dark. Fluorescence was recorded using an Olympus BX51WI upright microscope equipped with a 20× water immersion objective, 1 NA, and an Oregon-green filter cube (41026; Chroma). Fluorescence signals were focused on a photodiode and amplified with an Axopatch-200B patch clamp amplifier. Data were digitized at 5 kHz (Digidata 1550 A; Molecular Devices) and fluorescence signals were low-pass Bessel-filtered (Frequency Devices) at 100–200 Hz, digitized at 1 kHz, and collected using pClamp10 (Axon Instruments).

MD simulations and analysis

The homology models of the KCNQ3 channel transmembrane domain with S4 in the resting (down) state and active (up) state were created using the Swiss-model program (https://swissmodel.expasy.org/) based on the KCNQ1 channel model in the resting state70 and KCNQ2 channel in its activated (S4 up) state and the pore in the closed state71 (PDB: 7CR0), respectively. Each was embedded in POPC (palmitoyl oleoyl-phosphatidylcholine, ~2.5% phosphatidyl-4,5-bisphosphate (PIP2), and N-Arachidonoyl taurine (NAT–) using the membrane builder tool on CHARMM-GUI membrane builder72. All NAT– lipids were placed randomly beyond 10 Å from KCNQ3. A TIP3P water layer of 30 Å thickness on the upper bilayer and lower leaflet containing 150 mM of KCl was added in this system. CHARMM36 force field was used for protein73,74, POPC and PIP2 lipids75, KCl, and TIP3P water76. CHARMM general force fields (CGenFF)77 were used to prepare NAT– parameters. NAMD2.1378 was used for equilibrium and production simulations. The cutoff for calculating van der Waals interactions and short-range electrostatic interactions was set at 12 Å and force-switched at 10 Å. Long-range electrostatic interactions were calculated using the particle mesh Ewald algorithm79.

PUFA-KCNQ3 interaction analysis

Pairwise per-residue-based Generalized Born (GB) energy decomposition was performed using idecomp = 3 (adding 1-4 interactions to internal energy), igb = 5, saltcon = 0.150 options in MMPBSA.py (https://pubs.acs.org/doi/10.1021/ct300418h). The total pairwise energy is the sum of the van der Waals term, electrostatic term, generalized Born (GB) Polar solvation term, and Non-polar solvation term. 6 ns per snapshots were extracted from each trajectory for computing the interaction between each NAT lipid and one of 1048 protein residues. Three replicas of 600 ns runs were analyzed for the activated state. The resting state simulations were run with double length due to the weaker binding interactions. The sampling was determined to be sufficient when all replicates showed consistent ranking of the top three binding residues.

Electrophysiology data analysis

Data were analyzed with Clampfit 10 (Axon Instruments, Inc., Sunnyvale, CA), OriginPro 2021b (OriginLabs Northampton, MA), and Corel-DRAW Graphics Suite 2021 software. To determine the ionic conductance established by a given test voltage, a test voltage pulse was followed by a step to the fixed voltage of –40 mV (tail), and current was recorded following the step. To estimate the conductance g(V) activated at the end of the test pulse to voltage V, the current flowing after the hook was exponentially extrapolated to the time of the step and divided by the offset between –40 mV and the reversal potential. The conductance g(V) associated with different test voltages V in a given experiment was fitted by the relation:1 g(V)=A1+(A2 - A1)/1+exp((V1/2 - V)/κ)

where A1 and A2 are conductances that would be approached at extreme negative or positive voltages, respectively, V1/2 is the voltage that activates the conductance (A1 + A2)/2, and κ is the slope factor in mV. Due to the generally different numbers of expressed channels in different oocytes, we compare normalized conductance, G(V):2 G(V)=g(V)/A2

Fluorescence signals were corrected for bleaching and time-averaged over 10–40 ms intervals for analysis. The voltage dependence of fluorescence f(V) was analyzed and normalized (F(V)) using relations analogous to those for conductance (Eqs. 1 and 2).

To measure the effect of PUFAs, we first recorded the current vs. voltage (I-V) relationship in the control (without PUFAs) solution that we designated “before” in each Figure. For this protocol, cells were held at −80 mV followed by a step from −140 (or as indicated in each figure) to +40 mV (in 20 mV steps) followed by a subsequent voltage step to −40 mV (or 0 mV) to measure tail currents before returning to the −80 mV holding potential. We then continuously applied PUFAs to the chamber and oocytes were stepped every 15 s from −80 mV to 0 mV for 2 s followed by a voltage step to −40 mV before stepping to −80 mV to ensure that the PUFA effects on the current at 0 mV reached steady state (see Supplementary Fig. 4). We then used the same voltage-step protocol (as in control solution) to measure the current vs. voltage (I-V) relationship in the presence of PUFAs that we named “after PUFA” in each Figure.

The delta maximum conductance (ΔGmax) (or delta current at –60 mV (ΔI-60)) was calculated by taking the difference between the Gmax in PUFAs (GmaxPUFA) (or I-60 PUFA) and Gmax without PUFAs (Gmax0) (or I-60 0), divided by Gmax0 (or I-60 0), as:3 ΔGmax=(GmaxPUFA−Gmax0)/Gmax0

4 ΔI−60=(I-60PUFA−I-600)/I-600

To estimate the effect of mutations (mut) relative to the wild type (wt) and the effect of PUFAs on Gibbs free energy (∆∆G), the following relations were used:5 ΔΔG=Δ(zFV1/2)=−F(zwtV1/2wt−z(mut)V1/2(mut)).

6 ΔΔG=Δ(zFV1/2)=−F(zbeforeV1/2before−z(afterPUFA)V1/2(afterPUFA))

where F is Faraday’s constant, z (the slope; z = 25/s), and V1/2 (half-activation-voltage) were obtained from the mutation (mut), wild type (wt), control (before PUFA), and treated (after PUFA) G(V) curves. This analysis assumes a two-state model and tends to underestimate the z. The calculated ∆∆G should therefore be seen as an approximation. To analyze the effect of PUFAs on current and fluorescence kinetics we used T50%, which for opening (and closing) it was defined as the time it takes to reach 50% of the maximum (and minimum) current (or fluorescence) level at the end of the indicated depolarizing (opening or activated S4) and hyperpolarizing (closed or resting S4) pulse.

Statistics and reproducibility

All experiments were repeated 4 or more times from at least three batches of oocytes. Pairwise comparisons were achieved using ANOVA and Bonferroni’s post hoc test or Student’s t test as indicated in each figure. Data are represented as mean ± s.e.m (standard error of mean) and “n” represents the number of experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Peer Review File

Supplementary Information

Description of Additional Supplementary Materials

Supplementary Data 1

Reporting Summary

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-024-06873-4.

Acknowledgements

We thank Dr. Keneth Muller, Sara Alicante, and our team members for helpful comments and discussions. This work was supported by the National Institutes of Health, NINDS (1R01NS110847) to RB-S.

Author contributions

A.H.-P. conducted V.C.F. experiments and analyzed data; M.A.E., I.S., S.H., and Adl.C. conducted TEVC electrophysiology and analyzed data; M.E.P.-R., D.M.D., and M.D.-S. performed molecular biology and analyzed the data; M.E., Y.-C.L., and Y.L.L. performed molecular dynamic simulations; Y.L.L. and R.B.-S. prepared the figures; D.M.D., Y.L.L., and R.B.-S. wrote the manuscript; all authors edited the manuscript. R.B.-S. conceived and oversaw the study, and obtained funding for the project.

Peer review

Peer review information

Communications Biology thanks Geoffrey Abbott and Ferenc Papp for their contribution to the peer review of this work. Primary Handling Editors: Fereshteh Nugent and Benjamin Bessieres. A peer review file is available.

Data availability

Source data can be found in Supplementary Data 1.

Competing interests

Yun Lyna Luo is an Editorial Board Member for Communications Biology, but was not involved in the editorial review of, nor the decision to publish this article.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Michaela A. Edmond, Andy Hinojo-Perez.
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