Mechanistic insights into robust cardiac IKs potassium channel activation by aromatic polyunsaturated fatty acid analogues

Voltage-gated potassium (KV) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. KV channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore KV channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of KV channel activators with potential applications in the treatment of arrhythmogenic disorders such as Long QT Syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac IKs channel - a tetrameric potassium channel complex formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac IKs channel and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the IKs channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.

Interventions intended to restore KV channel function have strong therapeutic potential 27 in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise 28 a class of KV channel activators with potential applications in the treatment of 29 arrhythmogenic disorders such as Long QT Syndrome (LQTS). LQTS is caused by a 30 loss-of-function of the cardiac IKs channel -a tetrameric potassium channel complex 31 formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of 32 aromatic PUFA analogues that produce robust activation of the cardiac IKs channel and 33 a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We 34 determine the mechanisms through which tyrosine PUFA analogues exert strong 35 activating effects on the IKs channel by generating modified aromatic head groups 36 designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We 37 found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi 38 interactions, but instead do so through a combination of hydrogen bonding and ionic 39

interactions. 40
Introduction 41 The delayed rectifier potassium channel (IKs) underlies a critical repolarizing current that 42 determines the timing of the ventricular action potential 1 . The cardiac IKs current is 43 mediated by the association of the voltage gated K + channel KV7.1 α-subunit with the 44 KCNE1 -subunit 2-4 . The KV7.1 α-subunit consists of 6 transmembrane spanning 45 segments, denoted S1-S6 where S1-S4 form the voltage sensing domain (VSD) and 46 S5-S6 form the pore domain (PD) 5 . The S4 segment contains several positively charged 47 arginine residues that allow S4 to move outward, towards the extracellular side of the 48 membrane, when the membrane becomes depolarized 6 . This outward movement of the 49 S4 is transformed into pore opening as a result of conformational changes in the S4-S5 50 linker of KV7.1 7 . Co-expression of KCNE1 with KV7.1 imparts a more depolarized 51 voltage-dependence of activation, slower activation kinetics, and increased single 52 channel conductance compared to KV7.1 alone 8,9 . Loss-of-function mutations in the 53 cardiac IKs channel can lead to an arrhythmogenic disorder known as Long QT 54 Syndrome (LQTS), which predisposes individuals to ventricular fibrillation and sudden 55 cardiac death 10-12 . Current treatments for LQTS include pharmacological intervention 56 with β-blockers or surgical implantation of a cardioverter defibrillator 13 . However, 57 limitations of these treatments generate a need for novel therapeutic interventions to 58 treat LQTS. 59 60 Polyunsaturated fatty acids (PUFAs) are amphipathic molecules composed of a 61 charged hydrophilic head group and a long, polyunsaturated hydrophobic tail group 14 . It 62 is well-documented that PUFAs form a group of IKs channel activators that interact with 63 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint the channel voltage sensing domain (VSD) thus influencing the voltage dependence of 64 IKs channel activation [15][16][17] . PUFAs promote IKs channel activation through an 65 electrostatic interaction between the negative charge of the hydrophilic PUFA head and 66 positively charged arginine residues in the S4 segment of the IKs channel [17][18][19][20] This 67 electrostatic activation of the IKs channel is seen as a leftward shift in the voltage 68 dependence of IKs channel activation that leads to increases in IKs current. Recently, it 69 has been reported that PUFAs increase IKs current through two independent effects: 70 one on S4 (as described above) and one on the pore domain through an electrostatic 71 interaction with a positively charged lysine residue located in S6 (K326) 21 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint phe, 3,4,5F-NAL-phe, and 3F-NALT were synthesized similarly, with detailed 118 descriptions of the synthesis procedures for each compound provided in the 119 supplemental methods. PUFA analogues were kept at -20˚ C as 100 mM stock 120 solutions in ethanol except 4Br-NAL-phe, 4F-NAL-phe, 3,4,5F-NAL-phe, and 3F-NALT 121 where stock solutions were prepared as needed on the day of recording. Serial dilutions 122 of the different PUFAs were prepared from stocks to make 0.2 μM, 0.7 μM, 2.0 μM, 7.0 123 μM, and 20 μM concentrations in ND96 solutions (pH = 7.5). 124 125 Two-electrode voltage clamp (TEVC) 126 Xenopus laevis oocytes, co-expressing wild type KV7.1 and KCNE1, were recorded in 127 the two-electrode voltage-clamp (TEVC) configuration. Recording pipettes were filled 128 with 3 M KCl. The recording chamber was filled with ND96 (96 mM NaCl, 2 mM KCl, 1 129 mM MgCl2, 1.8 mM CaCl2, 5 mM Tricine; pH 9). Dilutions of PUFAs and PUFA 130 analogues were perfused into the recording chamber using the Rainin Dynamax 131 Peristaltic Pump (Model RP-1) (Rainin Instrument Co., Oakland, CA. USA). 132 Electrophysiological recordings were obtained using Clampex 10.3 software (Axon, 133 pClamp, Molecular Devices). During the application of PUFAs the membrane potential 134 was stepped every 30 sec from -80 mV to 0 mV for 5 seconds before stepping to -40 135 mV and back to -80 mV to ensure that the PUFA effects on the current at 0 mV reached 136 steady state (Fig. 1D). A voltage-step protocol was used to measure the current vs. 137 voltage (I-V) relationship before PUFA application and after the PUFA effects had 138 reached steady state for each concentration of PUFA. Cells were held at -80 mV 139 followed by a hyperpolarizing prepulse to -140 mV to make sure all channels are fully 140 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint closed. The voltage was then stepped from -100 to 60 mV (in 20 mV steps) followed by 141 a subsequent voltage step to -20 mV to measure tail currents before returning to the -80 142 mV holding potential. reported for each of the effects (I/I0, ΔV0.5, and Gmax) from the different PUFAs tested. In 161 some cases, there is variability in the V0.5 between batches of oocytes. In order to 162 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint correct for variability due to oocytes, when the V0.5 was greatly different than 20 mV in 163 control solution, we applied a correction in order to more accurately measure PUFA-164 induced IKs current increases. We subtracted the V0.5 (given by fitting the G-V with a 165 Boltzmann equation) by 20 mV and used the current measured at the resulting voltage. 166 The maximum conductance (Gmax) was calculated by taking the difference between the 167 maximum and minimum current values (using the G-V curve for each concentration) 168 and then normalizing to control solution (0 μM). Graphs plotting mean and standard 169 error of the mean (SEM) for I/I0, ΔV0.5, Gmax, and Km were generated using GraphPad  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made To measure the effects of the aromatic PUFA analogues on the cardiac IKs channel, we 182 expressed the IKs channel complex in Xenopus laevis oocytes (Fig. 1A). We co-injected 183 mRNA for the KV7.1 -subunit and the KCNE1 -subunit to achieve expression of 184 tetrameric IKs channels. Using two-electrode voltage-clamp recordings, we applied 185 depolarizing voltage steps to activate the IKs channel (   Figure   212 1-source data 1.

213
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. In addition, both NALT and Lin-tyrosine cause a significantly greater V0.5 compared to 230 NAL-phe and Lin-phe (p < 0.0001****; Fig. 2F). Together, these differences suggest that 231 cation-pi interactions are not the primary mechanism through which tyrosine PUFAs 232 activate the IKs channel. Rather, our data suggest that it is actually the presence of the 233 distal -OH group on the aromatic head group that is critical for the potent activation of 234 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023.

316
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint Aromatic PUFAs appear to activate the IKs channel in similar mechanisms as non-317 aromatic PUFAs 318 To better understand the mechanism of these superior activating aromatic PUFAs we 319 mutated residues previously shown to be important for non-aromatic PUFA activating 320 effects on IKs channels. The residue R231, located in the voltage sensor (S4) (Fig. 5A), 321 has been previously shown to be important for the V0.5 shifting effect of non-aromatic 322 PUFAs 22 . We tested Lin-tyr, the largest V0.5 shifting aromatic PUFA, on the IKs channel 323 with the mutation R231Q+Q234R to assess if R231 is also important for the aromatic 324 PUFA V0.5 shifting mechanism. The additional mutation Q234R is necessary to preserve 325 the voltage dependence of activation in IKs channels with the R231Q mutation 22,28,29 . 326 The V0.5 shifting effect of Lin-tyr was significantly decreased from -74.4mV  4.1 at 20 327 M in the wild-type (WT) IKs channel to -36.5mV  7.3 at 20 M with the R231Q+Q234R 328 mutation (p = 0.0021**; Fig. 5B-C). This reduction indicates that R231 contributes to 329 more than half of the voltage dependence shifting effect of Lin-tyr. The remaining shift is 330 most likely due to PUFA head group interactions with other nearby S4 charges such as 331

R228 and Q234R. 332
The residue K326, located near the pore, has been previously shown to be important for 333 the Gmax increasing effect of non-aromatic PUFAs 22 . We tested 3,4,5F NAL-phe, the 334 largest Gmax increasing aromatic PUFA, on the IKs channel with the mutation K326C to 335 assess if K326 is also important for the aromatic PUFA Gmax increasing mechanism 336 (Fig. 5D). The Gmax increasing effect of 3,4,5F NAL-phe was significantly decreased 337 from 2.4  0.4 at 20 M in the WT IKs channel to 1.22  0.2 at 20 M (p = 0.0287*; Fig.  338 5E-F). This reduction indicates that K326 is necessary for 3,4,5F NAL-phe's Gmax 339 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint increasing effect. Therefore, aromatic PUFA analogues modulate the IKs channel via two 340 independent interactions with residues in S4 (R231) and S6 (K326), consistent with the 341 previously described activation mechanisms of PUFAs on IKs channels (Fig. 5G). 342 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint the effects of NALT on mutated channels compared to the WT IKs channel (Fig.6A-B). 378 We found that S217A, Q220L, and S225A showed similar maximum shifts in voltage-379 dependent activation compared to the wild-type channel (WT + NALT: -56.1  3.6 mV; 380 S217A + NALT: -65.9  3.7 mV; Q220L + NALT: -59.5  11.1 mV; S225A + NALT: -52.4 381  3.7 mV at 20 M, ns); Fig. 6C-D). However, the T224V mutation significantly 382 attenuated the leftward shift in the voltage dependence of activation in response to 383 NALT application from -56.1  3.6 mV in WT channels to -32.1  7.0 at 20 M (p = 384 0.03*; Fig. 6D). To determine whether this effect was specific to compounds with the 385 ability to form hydrogen bonds we compared the effects of hydrogen-bonding NALT and 386 non-hydrogen-bonding NAL-phe on T224V mutant channels (Fig. 6E). In contrast to the 387 attenuation of the overall voltage shift observed when NALT was applied to the T224V, 388 there was no difference in the voltage-shifting effects of NAL-phe between the T224V 389 mutant and WT channels (WT + NAL-phe: -12.5  3.8 mV; T224V + NAL-phe: -7.8  2.1 390 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint mV at 20 M, ns (Fig. 6F-G). These data demonstrate that the T224V mutation only 391 reduces the efficacy of aromatic PUFAs that contain a hydrogen-bonding group like 392 tyrosine. As a result, we have identified a novel interaction between the S3-S4 loop 393 residue T224 and hydrogen bonding moieties of aromatic PUFA head groups (Fig. 6H).  surface potential through addition of fluorine atoms to the NAL-phe head group (3,4,5F-445 NAL-phe), therefore, is expected to reduce the efficacy of 3,4,5F-NAL-phe in 446 comparison to NAL-phe alone. However, we find the opposite when we apply 3,4,5F-447 NAL-phe to the cardiac IKs channel, and see that 3,4,5F-NAL-phe is a more potent 448 activator of the IKs channel compared to NAL-phe alone. Together, these data suggest 449 that cation-pi interactions are not the primary mechanism through which these aromatic 450 PUFA analogues activate the cardiac IKs channel. 451

452
When we look at several fluorinated and brominated phenylalanine PUFA analogues, 453 we find specifically that 3,4,5F-NAL-phe has significantly greater effects on I/I0 and V0.5 454 compared to NAL-phe alone. While not statistically significant, 4Br-, 4F-, and 3,4,5F-455 NAL-phe also lead to some of the most consistent increases in Gmax among the PUFA 456 analogues tested in this work, with each of these compounds leading to a two-fold 457 increase in Gmax. These data suggest that aromatic PUFA analogues with highly (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Site 2 at K326 in the S6 segment 21 . We here show that the superior-activating aromatic 490 PUFAs also act on these sites in S4 and S6. To do this we selected the best V0.5 shifting 491 aromatic PUFA (Lin-tyr) to test on the IKs channel with the S4 mutation R231Q. 492 Additionally, we selected the best Gmax increasing aromatic PUFA (3,4,5F-NAL-phe) to 493 test on the IKs channel with S6 mutation K326C. The mutation R231Q decreases the 494 V0.5 shifting effect of Lin-tyr by half, indicating that Lin-tyr is shifting the voltage 495 dependence by creating an electrostatic interaction with the positive charges on the 496 voltage sensor. Conversely, the mutation K326C almost completely removed the Gmax 497 increasing effect of 3,4,5F-NAL-phe. We therefore propose that the increased effects of 498 the aromatic PUFAs, compared to non-aromatic PUFAs, are due to the additional 499 hydrogen bonding in Site 1 and electrostatic interactions in Site 2 to better anchor them 500 in these binding sites to increase their effects (Fig. 5G). As mentioned above, we also 501 show that the aromatic rings have the potential to be modified to give preferential effects 502 on either the IKs channel voltage sensor or channel pore. 503

504
Our experiments with NAL-Phe and 3F-NALT show that the hydrogen bonding capacity 505 of the -OH on the tyrosine of NALT is necessary for it to have superior voltage 506 dependence shifting effect. We further discovered the specific details of the hydrogen 507 bond interactions between this -OH group of NALT and the S3-S4 loop of the IKs 508 channel. We mutated all residues capable of hydrogen bonding in the S3-S4 loop, 509 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint removing their ability to hydrogen bond and tested if this changed the NALT voltage 510 dependence shifting effect. The voltage dependence of mutations S217A, Q220L, and 511 S225A was shifted to the same degree by NALT as the wild type IKs channel. However, 512 the voltage dependence of mutation T224V was shifted significantly less than the WT IKs 513 channels. This shows that the -OH group on the tyrosine of NALT hydrogen bonds with 514 T224V thereby improving the PUFA's ability to shift the voltage dependence. This 515 hydrogen bond interaction between PUFAs and the 3-4 loop of the IKs channel is a novel 516 mechanism to increase the effect of PUFAs to activate the IKs channel. These data 517 suggest that drugs designed to target this interaction would be more effective at shifting 518 IKs channel voltage dependence. 519 520 Overall, our findings suggest that different aromatic PUFA analogs not only increase 521 PUFA efficacy on activating the IKs channel, but their specific effects on IKs function can 522 be modulated independently, either increasing the maximal conductance or voltage-523 shifting effect. This novel mechanistic understanding of how aromatic PUFAs have 524 these increased effects on the IKs channel may help to aid drug development for Long 525 QT Syndrome. This data provides insight into how PUFA activation of the IKs channel 526 can be both increased and tailored to specific IKs channel deficiencies, such as shifts in 527 voltage dependence and decreases in maximal conductance. 528 529 530 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023.  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523777 doi: bioRxiv preprint