Bohannon BM et al. (2020), Polyunsaturated fatty acids produce a range of ...

XB-ART-58191

J Gen Physiol 2020 Feb 03;1522:. doi: 10.1085/jgp.201912396.

Show Gene links Show Anatomy links

Polyunsaturated fatty acids produce a range of activators for heterogeneous IKs channel dysfunction.

Bohannon BM , Wu X , Wu X , Perez ME , Liin SI , Larsson HP .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 31865382
???displayArticle.pmcLink??? PMC7062506
???displayArticle.link??? J Gen Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis

???attribute.lit??? ???displayArticles.show???

	Figure 1. Illustration of the lipoelectric mechanism and measured effects on the cardiac IKs channel. (A) Schematic side view (left) of the IKs channel with S4 in green. Illustration (right) of the electrostatic interaction of a PUFA analogue (orange) with the voltage sensor (green) of the cardiac IKs channel, which leads to potentiation of upward S4 movement. (B) Schematic top view (left) of the IKs channel with Kv7.1 in blue (light blue, pore domain; dark blue, voltage-sensing domain) and KCNE1 in purple. Illustration (right) of the electrostatic interaction of a PUFA analogue (orange) with the positively charged lysine residue K326 in the S6 segment of the cardiac IKs channel, which leads to an increase in the Gmax of the IKs channel. (C) Activation protocol for the cardiac IKs channel using two-electrode voltage clamp and raw current traces in 0 µM PUFA analogue (left) and 20 µM PUFA analogue (right), with arrows indicating tail currents. Red trace occurs at 20 mV for visualization of PUFA-induced increases in current. (D) Representative current versus voltage relationship in 0 µM (black line) and 20 µM PUFA (blue line) highlighting an increase in I/I0 at 0 mV, leftward shift in the V0.5, and increase in Gmax denoted by arrows.
	Figure S1. Current versus voltage relationship between pH 7.5 and pH 9.0. Current–voltage relationship of IKs channel in pH 7.5 (black squares; mean ± SEM; n = 4) and pH 9.0 (red circles; mean ± SEM; n = 4).
	Figure S2. Reduction of IKs current by the application of DHA taurine is not intrinsically related to the taurine head group alone. (A) Structure of taurine and raw current traces measured in 0 µM taurine (left) and 100 µM taurine (right). Red trace shows currents at 20 mV for visualization of no effects of taurine on the voltage dependence. (B) Current versus voltage relationship in control (0 µM taurine) and following the addition of 100 µM taurine (mean ± SEM; n = 4).
	Figure S3. Residues in the voltage sensor and pore are important for electrostatic activation of the cardiac IKs channel. (A) Current–voltage relationship for Kv7.1 R231Q Q234R + KCNE1 with the application of lin-glycine (mean ± SEM; n = 4). (B-D) Dose-dependent effects of lin-glycine on Kv7.1 R231Q Q234R + KCNE1 (black squares) and the wild- type IKs channel (red dashed line) on (B) I/I0, (C) ΔV0.5, and (D) Gmax. (E) Current-voltage relationship for Kv7.1 K326Q + KCNE1 with the application of lin-glycine (mean ± SEM; n = 4). (F-H) Dose-dependent effects of lin-glycine on Kv7.1 K326Q + KCNE1 (black squares) and the wild-type IKs channel (red dashed line) on (F) I/I0, (G) ΔV0.5, (H) Gmax.
	Figure S4. Effects of lin-AP3 on IKs activation at pH 9.0. (A-C) Dose-dependent effects of lin-AP3 on (A) IKs current (I/I0), (B) IKs voltage dependence (ΔV0.5), and (C) IKs Gmax at pH 9.0 (mean ± SEM at maximal concentration; n = 3).
	Figure 2. Lin-taurine produces the most potent activation of the IKs channel compared with lin-glycine and linoleic acid. (A–C) Structure of and raw current traces measured in 0 µM (left) and 20 µM (right) (A) linoleic acid, (B) lin-glycine, and (C) lin-taurine. Red trace shows currents at 20 mV for visualization of PUFA-induced effects on current. (D) Dose-dependent effects of linoleic acid (n = 5), lin-glycine (n = 4), and lin-taurine (n = 3) on IKs current (I/I0; mean ± SEM at maximal concentration). (E) Statistical differences on I/I0 effects (I/I0 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (F) Dose-dependent effects of linoleic acid, lin-glycine, and lin-taurine on IKs voltage dependence (ΔV0.5). (G) Statistical differences on ΔV0.5 effects (ΔV0.5 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (H) Dose-dependent effects of linoleic acid, lin-glycine, and lin-taurine on IKs Gmax. (I) Statistical differences on Gmax effects (Gmax fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.05; , P < 0.001; **, P < 0.0001.
	Figure 3. Increasing the length of the lin-glycine head group alters the pKa and reduces the activating effect on the IKs channel. (A and B) Structure of (A) lin-glycine with the addition of one carbon in the head group (lin-glycine+1C) and (B) lin-glycine with the addition of two carbons in the head group (lin-glycine+2C). (C–H) Dose-dependent effects of lin-glycine (n = 4), lin-glycine+1C (n = 3), and lin-glycine+2C (n = 3) on (C) IKs current (I/I0) at pH 7.5, (D) IKs voltage dependence (ΔV0.5) at pH 7.5, (E) IKs Gmax at pH 7.5, (F) I/I0 at pH 9.0, (G) ΔV0.5 at pH 9.0, and (H) Gmax at pH 9.0 (mean ± SEM at maximal concentration). (I–K) Statistical differences at 20 µM on (I) I/I0 effect, (J) ΔV0.5 effect, and (K) Gmax effect measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.05; , P < 0.01; **, P < 0.001. ns, not significant.
	Figure 4. Increasing the number of potentially charged moieties of the PUFA head group does not improve PUFA-induced IKs activation. (A–C) Structure of and raw current traces measured in 0 µM (left) and 20 µM (right) (A) lin-aspartate, (B) lin-cysteic acid, and (C) lin-AP3. Red trace shows currents at 20 mV for visualization of PUFA-induced effects on current. (D–F) Dose-dependent effects of lin-glycine (n = 4), lin-taurine (n = 3), lin-aspartate (n = 4), lin-cysteic acid (n = 5), and lin-AP3 (n = 3) on (D) IKs current (I/I0), (E) IKs voltage dependence (ΔV0.5), and (F) IKs Gmax (mean ± SEM at maximal concentration). (G-I) Statistical differences at 20 µM on (G) I/I0 effect, (H) ΔV0.5 effect, and (I) Gmax effect measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.05; , P < 0.01; **, P < 0.001. ns, not significant.
	Figure 5. DHA-taurine at 7 µM produces the most potent activation of the IKs channel compared with DHA-glycine and DHA at 20 µM. (A–C) Structure of and raw current traces measured in 0 µM (left) and 20 µM (right) (A) DHA, (B) DHA-glycine, and (C) DHA-taurine, 0 µM (left) and 7 µM (right). We report effects of DHA-taurine at 7 µM due to an unclear reduction in current caused by the application of 20 µM. Red trace shows currents at 20 mV for visualization of PUFA-induced effects on current. (D) Dose-dependent effects of DHA (n = 4), DHA-glycine (n = 4), and DHA-taurine (n = 3) on IKs current (I/I0; mean ± SEM at maximal concentration). (E) Statistical differences on I/I0 effects (I/I0 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (F) Dose-dependent effects of DHA, DHA-glycine, and DHA-taurine on IKs voltage dependence (ΔV0.5). (G) Statistical differences on ΔV0.5 effects (ΔV0.5 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (H) Dose-dependent effects of DHA, DHA-glycine, and DHA-taurine on IKs Gmax. (I) Statistical differences on Gmax effects (Gmax fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.05; ***, P < 0.0001. ns, not significant.
	Figure 6. Pin-taurine produces the most potent activation of the IKs channel compared with pin-glycine and pinolenic acid. (A–C) Structure of and raw current traces measured in 0 µM (left) and 20 µM (right) (A) pinolenic acid, (B) pin-glycine, and (C) pin-taurine. Red trace shows currents at 20 mV for visualization of PUFA-induced effects on current. (D) Dose-dependent effects of pinolenic acid (n = 3), pin-glycine (n = 3), and pin-taurine (n = 4) on IKs current (I/I0; mean ± SEM at maximal concentration). (E) Statistical differences on I/I0 effects (I/I0 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (F) Dose-dependent effects of pinolenic acid, pin-glycine, and pin-taurine on IKs voltage dependence (ΔV0.5). (G) Statistical differences on ΔV0.5 effects (ΔV0.5 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (H) Dose-dependent effects of pinolenic acid, pin-glycine, and pin-taurine on IKs Gmax. (I) Statistical differences on Gmax effects (Gmax fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.01; , P < 0.001; ***, P < 0.0001. ns, not significant.
	Figure 7. Comparison of effects by glycine head groups and taurine head groups on IKs current (I/I0), voltage dependence (ΔV0.5), and Gmax. (A–C) Dose-dependent effects of DHA-glycine (n = 4), lin-glycine (n = 4), pin-glycine (n = 3), DHA-taurine (n = 3), lin-taurine (n = 3), and pin-taurine (n = 4) on (A) IKs current (I/I0), (B) IKs voltage dependence (ΔV0.5), and (C) IKs Gmax (mean ± SEM at maximal concentration). Gray open square is for DHA-taurine at 20 μM (not included in fit).
	Figure 8. Hierarchical cluster analysis and heat map demonstrate that taurine head groups are most similar in their voltage-shifting effects and glycine head groups are most similar in their effects on Gmax. The dendrogram displays groupings of PUFAs and PUFA analogues according to similarity of their effects. The heat map displays the magnitude of the effects, with warmer colors representing PUFAs and PUFA analogues that have larger relative effects (closer to 1.0) on I/I0, Gmax, and ΔV0.5 and cooler colors representing PUFAs and PUFA analogues with smaller relative effects (closer to 0.0).
	Figure 9. Lin-glycine in combination with physiological concentrations of monounsaturated and saturated fatty acids and albumin promotes the activation of the IKs channel. (A) Raw current traces measured in control ND96 (left) and in the presence of 0.1 mM albumin + 0.2 mM lin-glycine/0.2 mM oleic acid/0.2 mM stearic acid (Fatty Acids; right). Red trace shows currents at 20 mV for visualization of PUFA-induced effects on current. (B) Current-voltage relationship of cells in control ND96 (black squares) and in 0.1 mM albumin + 0.2 mM lin-glycine/0.2 mM oleic acid/0.2 mM stearic acid (fatty acids [FAs]; red circles; mean ± SEM; n = 4). (C) Statistical differences on I/I0 effects (I/I0 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (D) Statistical differences on ΔV0.5 effects (ΔV0.5 fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. (E) Statistical differences on Gmax effects (Gmax fitted from the dose–response curve) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. , P < 0.01; *, P < 0.001. ns, not significant.
	Figure 10. PUFA analogues lin-glycine and lin-taurine rescue LQT1-associated loss-of-function mutation, V215M. (A) Topology of Kv7.1 and KCNE1 with location of V215M indicated. (B) Current–voltage relationship of the wild-type IKs channel (black squares; mean ± SEM; n = 4), Kv7.1 V215M + KCNE1 (red circles; mean ± SEM; n = 3), Kv7.1 V215M + KCNE1 with lin-glycine (green triangles; mean ± SEM; n = 3), and Kv7.1 V215M + KCNE1 with lin-taurine (blue triangles; mean ± SEM; n = 3). (C) Statistical differences on the voltage dependence (V0.5) effects (V0.5 fitted using the Boltzmann equation) measured by one-way ANOVA followed by Tukey’s HSD post hoc analysis. ****, P < 0.0001. Error bars represent SEM.

References [+] :

Abdelmagid, Comprehensive profiling of plasma fatty acid concentrations in young healthy Canadian adults. 2015, Pubmed