Repolarization and termination of the ventricular cardiac action potential is highly dependent on the activation of the slow delayed-rectifier potassium IKs channel. Disruption of the IKs current leads to the most common form of congenital long QT syndrome (LQTS), a disease that predisposes patients to ventricular arrhythmias and sudden cardiac death. We previously demonstrated that polyunsaturated fatty acid (PUFA) analogues increase outward K+ current in wild type and LQTS-causing mutant IKs channels. Our group has also demonstrated the necessity of a negatively charged PUFA head group for potent activation of the IKs channel through electrostatic interactions with the voltage-sensing and pore domains. Here, we test whether the efficacy of the PUFAs can be tuned by the presence of different functional groups in the PUFA head, thereby altering the electrostatic interactions of the PUFA head group with the voltage sensor or the pore. We show that PUFA analogues with taurine and cysteic head groups produced the most potent activation of IKs channels, largely by shifting the voltage dependence of activation. In comparison, the effect on voltage dependence of PUFA analogues with glycine and aspartate head groups was half that of the taurine and cysteic head groups, whereas the effect on maximal conductance was similar. Increasing the number of potentially negatively charged moieties did not enhance the effects of the PUFA on the IKs channel. Our results show that one can tune the efficacy of PUFAs on IKs channels by altering the pKa of the PUFA head group. Different PUFAs with different efficacy on IKs channels could be developed into more personalized treatments for LQTS patients with a varying degree of IKs channel dysfunction.

Polyunsaturated fatty acids with double bonds closer to the head group had higher apparent affinity for I channels and increased I current more; shifting the bonds further away from the head group reduced apparent binding affinity for and effects on the I current. Interestingly, we found that ω-6 and ω-9 PUFAs, with the first double bond closer to the head group, left-shifted the voltage dependence of activation the most. These results allow for informed design of new therapeutics targeting I channels in Long QT Syndrome.

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

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20The cardiac ventricular action potential depends on several voltage-gated ion channels, 21 including Nav, Cav, and Kv channels. Mutations in these channels can cause Long QT 22 Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac 23 death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for 24 LQTS because they are modulators of voltage-gated ion channels. Here we 25 demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion 26 channels involved in the ventricular action potential. The effects of specific PUFA 27 analogues range from selective for a specific ion channel to broadly modulating all three 28 cardiac ion channels (NaV, CaL, and IKs). In addition, PUFA analogues do not modulate 29 these channels through a shared mechanism. Our data suggest that different PUFA 30 analogues could be tailored towards specific forms of LQTS, which are caused by 31 mutations in distinct cardiac ion channels, and thus restore a normal ventricular action 32 potential. 34Polyunsaturated fatty acids (PUFAs) are amphipathic molecules that have been 82 suggested to possess antiarrhythmic effects (29, 30). PUFAs are characterized by 83 having a long hydrocarbon tail with two or more double bonds, as well as having a 84 charged, hydrophilic head group (31). PUFAs, such as DHA and EPA, have been 85 shown to prevent cardiac arrhythmias in animal models and cultured cardiomyocytes by 86 inhibiting the activity of Nav and Cav channels (30,(32)(33)(34). Specifically, DHA and EPA 87 are thought to bind to discrete sites on the channel protein to stabilize the inactivated 88 states of the Nav and Cav channels (32, 35). Since the voltage sensors of Nav and Cav 89 channels are relatively homologous, it has been suggested that PUFAs act on the 90 voltage-sensing S4 segments that control inactivation in these channels (30, 32). Our 91 group has demonstrated that PUFAs and PUFA analogues also modulate the activity of 92 the Kv7.1/KCNE1 channel and work to promote voltage-dependent activation of the IKs 93 current (36-38). The mechanism through which PUFAs promote Kv7.1/KCNE1 94 activation is referred to as the lipoelectric hypothesis which involves the following: 1) the 95 PUFA molecule integrates into the membrane via its hydrocarbon tail and 2) the 96 negatively charged PUFA head group electrostatically attracts the positively charged S4 97 segment and facilitates the outward movement of S4, promoting IKs channel activation 98 (36). Our group has also demonstrated that PUFAs and modified PUFAs exert a second 99 effect on the pore of Kv7.1 through an additional electrostatic interaction with a lysine 100 residue (K326) in the S6 segment (39). This electrostatic interaction between the 101 negatively charged PUFA head group and K326 leads to an increase in maximal 102 conductance of the channel (Gmax) (39).103 104 6Some groups have suggested that PUFAs could modify Nav channels by causing a 105 leftward shift in voltage dependent inactivation through...