Ma R and Lewis A (2020), Spadin Selectively Antagonizes Arachidonic Acid...

XB-ART-56906

Front Pharmacol 2020 Apr 07;11:434. doi: 10.3389/fphar.2020.00434.

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Spadin Selectively Antagonizes Arachidonic Acid Activation of TREK-1 Channels.

Ma R , Lewis A .

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TREK-1 channel activity is a critical regulator of neuronal, cardiac, and smooth muscle physiology and pathology. The antidepressant peptide, spadin, has been proposed to be a TREK-1-specific blocker. Here we sought to examine the mechanism of action underlying spadin inhibition of TREK-1 channels. Heterologous expression in Xenopus laevis oocytes and electrophysiological analysis using two-electrode voltage clamp in standard bath solutions was used to characterize the pharmacological profile of wild-type and mutant murine TREK-1 and TREK-2 channels using previously established human K2P activators; arachidonic acid (AA), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), BL-1249, and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and inhibitors; spadin and barium (Ba2+). Mouse TREK-1 and TREK-2 channel currents were both significantly increased by AA, BL-1249, and CDC, similar to their human homologs. Under basal conditions, both TREK-1 and TREK-2 currents were insensitive to application of spadin, but could be blocked by Ba2+. Spadin did not significantly inhibit either TREK-1 or TREK-2 currents either chemically activated by AA, BL-1249, or CDC, or structurally activated via a gating mutation. However, pre-exposure to spadin significantly perturbed the subsequent activation of TREK-1 currents by AA, but not TREK-2. Furthermore, spadin was unable to prevent activation of TREK-1 by BL-1249, CDC, or the related bioactive lipid, DHA. Spadin specifically antagonizes the activation of TREK-1 channels by AA, likely via an allosteric mechanism. Lack of intrinsic activity may explain the absence of clinical side effects during antidepressant therapy.

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	Figure 1. Mouse TREK-1 and TREK-2 channels are biophysically and pharmacologically comparable to their human counterparts. Exemplar current traces of mTREK-1 (A) and mTREK-2 (D) elicited by voltage pulses from −120 to +60 mV in 10 mV steps, for 500 ms from a holding potential of −80 mV (inset), in both asymmetric K+ solutions (4 mM) or symmetric K+ solutions (140 mM). In each panel, scale bars represent 2 μA and 100 ms. The zero current level is indicated by red dashed line. (B, E) Mean current-voltage (I-V) relationships taken from steady-state currents in bath solution containing low (asymmetric) K+ (4 mM, black squares) or high (symmetric) K+ (140 mM, open squares) for (B) mTREK-1 (n = 7) and (E) mTREK-2 (n = 8). (C, F) Reversal potential (EREV) plotted as a function of [K+]o (mean ± SEM) for (C) mTREK-1 and (F) mTREK-2 currents. Data points are fitted with a linear relationship (red line). Dashed line represents Nernstian theoretical linear relationship of equilibrium potential for K+ (EK) with changing [K+]o as calculated using the Nernst equation, assuming [K+]i of 140 mM (slope = −58.2 mV). (G) Chemical structures of three main activators of TREK channels. (H, I) Bar graphs showing effects on mean (± SEM) normalized mTREK-1 (H) and mTREK-2 (I) currents measured at 0 mV (in %) after application of either 10 μM AA, 1 μM BL-1249 (mTREK-1), 3 μM BL-1249 (mTREK-2), 10 μM CDC, or 1 mM Ba2+. Dashed line indicates control current level. All data represents n > 6 oocytes. *p < 0.001, **p < 0.0001, Student's paired t-test conducted on raw data.
	Figure 2. Spadin specifically antagonizes AA-activation of TREK-1 channels. (A, C) Exemplar time-courses of mTREK-1 currents measured at 0 mV recorded from oocytes by TEVC, under different bath conditions; control (4K, black), 1 μM spadin alone (Sp, red), 10 μM AA alone (AA, green), or 1 μM spadin plus 10 μM AA (Sp +AA, white) as indicated on bar. (B, D) Mean current-voltage relationships of experiments from A and C respectively (n = 8–12). Numbers in brackets refer to the order of application of compounds, mirroring respective time-courses. (E, F) Mean bar graphs showing the effect of spadin on mTREK-1 current activation by (E) BL-1249 and (F) CDC. mTREK-1 current amplitudes were recorded at 0 mV by TEVC in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), 1 μM spadin plus indicated activator (white) and activators alone; 1 μM BL-1249 (orange) or 10 μM CDC (blue). (G, H) Mean bar graphs showing the effect of spadin on mTREK-1 currents amplitudes recorded at 0 mV by TEVC in the presence of DHA. (G) Mean mTREK-1 currents recorded in control bath conditions (4K, black), and following pre-activation with 10 μM DHA (green) and when supplemented with 1 μM spadin (white). (H) Mean mTREK-1 currents recorded in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), supplementation with 10 μM DHA (white) or 10 uM DHA alone (green). Data are presented as mean ± SEM, n = 10 for both. n.s, not significant (p > 0.05), ****p < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test.
	Figure 3. Spadin does not antagonize AA-activation of mTREK-2 channels. (A, C) Exemplar time-courses of mTREK-2 currents measured at 0 mV recorded from oocytes by TEVC, under different bath conditions; control 4K (black), 1 μM spadin alone (red), 10 μM AA alone (green) or 1 μM spadin plus 10 μM AA (white) as indicated on bar. (B, D) Mean current-voltage relationships of experiments from A and C respectively (n = 7-9). Numbers in brackets refer to the order of application of compounds, mirroring respective time-courses. (E, F) Mean bar graphs showing the effect of spadin on mTREK-2 current activation by (E) BL-1249 (F) CDC. mTREK-2 current amplitudes were recorded at 0 mV by TEVC in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), 1 μM spadin plus indicated activator (white) and activators alone; 3 μM BL-1249 (orange) or 10 μM CDC (blue). Data are presented as mean ± SEM, n = 8-10. n.s, not significant (p > 0.05), p < 0.05, ***p < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test.
	Figure 4. Ba2+ blocks AA and BL-1249 activated of mTREK-1 and mTREK-2 channels. Bar graphs displaying the effects of barium ion (Ba2+) block of (A) AA-activated mTREK-1, (B) AA-activated mTREK-2, (C) BL-1249 activated mTREK-1, and (D) BL-1249 activated mTREK-2 channel currents. mTREK-1 and mTREK-2 channel currents were recorded from Xenopus laevis oocytes by TEVC. (A, C) Mean currents for mTREK-1 at 0 mV showing the effects of 1 mM Ba2+ after pre-activation by 10 μM AA (A, n = 8) or 1 μM BL-1249 (C, n = 10). (B, D) Mean currents for mTREK-2 at 0 mV showing the effects of 1 mM Ba2+ after pre-activation with 10 μM AA (B, n = 6) or 3 μM BL-1249 (D, n = 9) activation. Data are presented as mean ± SEM. p < 0.05, p <0.01, p < 0.001, **P < 0.0001, Student's paired t-test.
	Figure 5. Gating mutation in TM4 reveals spadin is unable to inhibit hyper-active mTREK-1 channels. (A) left panel, structural diagram of mTREK-1 showing position of Y284A gating mutation, right panel, sequence alignment of distal pore-lining transmembrane domains (TM4) of mTREK-1 (NP_034737), hTREK-2 (NP_612191), KCNK0 (AAC69250), (TM6) Shaker (CAA29917), and (TM2) KcsA (POA333.1). Highlighted in bold are the tyrosine residue of mTREK-1 (Y284) and the analogous tyrosine in hTREK-2 (Y315). Mutation of Y315 to alanine significantly reduces norfluoxetine activity in human TREK-2 (Mcclenaghan et al., 2016). * Conserved glycine hinge residue (boxed). Mutation to this residue is known to promote stabilization of the open state across the potassium channels listed. (B, C) Mean bar graphs of chimera channel currents recorded at 0 mV by TEVC in control bath conditions (4K, black), and following treatment with (B) 10 uM AA (n = 9, green) or (C) 1 μM spadin (n = 7, red). Data are presented as mean ± SEM. p >0.05, not significant (n.s).

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	Figure 1. Mouse TREK-1 and TREK-2 channels are biophysically and pharmacologically comparable to their human counterparts. Exemplar current traces of mTREK-1 (A) and mTREK-2 (D) elicited by voltage pulses from −120 to +60 mV in 10 mV steps, for 500 ms from a holding potential of −80 mV (inset), in both asymmetric K+ solutions (4 mM) or symmetric K+ solutions (140 mM). In each panel, scale bars represent 2 μA and 100 ms. The zero current level is indicated by red dashed line. (B, E) Mean current-voltage (I-V) relationships taken from steady-state currents in bath solution containing low (asymmetric) K+ (4 mM, black squares) or high (symmetric) K+ (140 mM, open squares) for (B) mTREK-1 (n = 7) and (E) mTREK-2 (n = 8). (C, F) Reversal potential (EREV) plotted as a function of [K+]o (mean ± SEM) for (C) mTREK-1 and (F) mTREK-2 currents. Data points are fitted with a linear relationship (red line). Dashed line represents Nernstian theoretical linear relationship of equilibrium potential for K+ (EK) with changing [K+]o as calculated using the Nernst equation, assuming [K+]i of 140 mM (slope = −58.2 mV). (G) Chemical structures of three main activators of TREK channels. (H, I) Bar graphs showing effects on mean (± SEM) normalized mTREK-1 (H) and mTREK-2 (I) currents measured at 0 mV (in %) after application of either 10 μM AA, 1 μM BL-1249 (mTREK-1), 3 μM BL-1249 (mTREK-2), 10 μM CDC, or 1 mM Ba2+. Dashed line indicates control current level. All data represents n > 6 oocytes. *p < 0.001, **p < 0.0001, Student's paired t-test conducted on raw data.
	Figure 2. Spadin specifically antagonizes AA-activation of TREK-1 channels. (A, C) Exemplar time-courses of mTREK-1 currents measured at 0 mV recorded from oocytes by TEVC, under different bath conditions; control (4K, black), 1 μM spadin alone (Sp, red), 10 μM AA alone (AA, green), or 1 μM spadin plus 10 μM AA (Sp +AA, white) as indicated on bar. (B, D) Mean current-voltage relationships of experiments from A and C respectively (n = 8–12). Numbers in brackets refer to the order of application of compounds, mirroring respective time-courses. (E, F) Mean bar graphs showing the effect of spadin on mTREK-1 current activation by (E) BL-1249 and (F) CDC. mTREK-1 current amplitudes were recorded at 0 mV by TEVC in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), 1 μM spadin plus indicated activator (white) and activators alone; 1 μM BL-1249 (orange) or 10 μM CDC (blue). (G, H) Mean bar graphs showing the effect of spadin on mTREK-1 currents amplitudes recorded at 0 mV by TEVC in the presence of DHA. (G) Mean mTREK-1 currents recorded in control bath conditions (4K, black), and following pre-activation with 10 μM DHA (green) and when supplemented with 1 μM spadin (white). (H) Mean mTREK-1 currents recorded in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), supplementation with 10 μM DHA (white) or 10 uM DHA alone (green). Data are presented as mean ± SEM, n = 10 for both. n.s, not significant (p > 0.05), ****p < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test.
	Figure 3. Spadin does not antagonize AA-activation of mTREK-2 channels. (A, C) Exemplar time-courses of mTREK-2 currents measured at 0 mV recorded from oocytes by TEVC, under different bath conditions; control 4K (black), 1 μM spadin alone (red), 10 μM AA alone (green) or 1 μM spadin plus 10 μM AA (white) as indicated on bar. (B, D) Mean current-voltage relationships of experiments from A and C respectively (n = 7-9). Numbers in brackets refer to the order of application of compounds, mirroring respective time-courses. (E, F) Mean bar graphs showing the effect of spadin on mTREK-2 current activation by (E) BL-1249 (F) CDC. mTREK-2 current amplitudes were recorded at 0 mV by TEVC in control bath conditions (4K, black), and following pre-treatment with 1 μM spadin alone (red), 1 μM spadin plus indicated activator (white) and activators alone; 3 μM BL-1249 (orange) or 10 μM CDC (blue). Data are presented as mean ± SEM, n = 8-10. n.s, not significant (p > 0.05), p < 0.05, ***p < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test.
	Figure 4. Ba2+ blocks AA and BL-1249 activated of mTREK-1 and mTREK-2 channels. Bar graphs displaying the effects of barium ion (Ba2+) block of (A) AA-activated mTREK-1, (B) AA-activated mTREK-2, (C) BL-1249 activated mTREK-1, and (D) BL-1249 activated mTREK-2 channel currents. mTREK-1 and mTREK-2 channel currents were recorded from Xenopus laevis oocytes by TEVC. (A, C) Mean currents for mTREK-1 at 0 mV showing the effects of 1 mM Ba2+ after pre-activation by 10 μM AA (A, n = 8) or 1 μM BL-1249 (C, n = 10). (B, D) Mean currents for mTREK-2 at 0 mV showing the effects of 1 mM Ba2+ after pre-activation with 10 μM AA (B, n = 6) or 3 μM BL-1249 (D, n = 9) activation. Data are presented as mean ± SEM. p < 0.05, p <0.01, p < 0.001, **P < 0.0001, Student's paired t-test.
	Figure 5. Gating mutation in TM4 reveals spadin is unable to inhibit hyper-active mTREK-1 channels. (A) left panel, structural diagram of mTREK-1 showing position of Y284A gating mutation, right panel, sequence alignment of distal pore-lining transmembrane domains (TM4) of mTREK-1 (NP_034737), hTREK-2 (NP_612191), KCNK0 (AAC69250), (TM6) Shaker (CAA29917), and (TM2) KcsA (POA333.1). Highlighted in bold are the tyrosine residue of mTREK-1 (Y284) and the analogous tyrosine in hTREK-2 (Y315). Mutation of Y315 to alanine significantly reduces norfluoxetine activity in human TREK-2 (Mcclenaghan et al., 2016). * Conserved glycine hinge residue (boxed). Mutation to this residue is known to promote stabilization of the open state across the potassium channels listed. (B, C) Mean bar graphs of chimera channel currents recorded at 0 mV by TEVC in control bath conditions (4K, black), and following treatment with (B) 10 uM AA (n = 9, green) or (C) 1 μM spadin (n = 7, red). Data are presented as mean ± SEM. p >0.05, not significant (n.s).