Debreczeni D , Baukál D , Pergel E , Veres I , Czirják G .

	Figure 1. Schematic membrane topology of human TRESK. TRESK functions as a homodimer of subunits. In this figure, one subunit is illustrated, which follows the general 4TMS/2P molecular architecture of K2P channels; it possesses four TMS1-4 and two pore domains (P1-2). However, the proportions of intracellular regions are unique in TRESK. The long intracellular loop (121 amino acids) contains the known regulatory elements. For simplicity, only calcineurin and its binding sites (the PQIIIS and LQLP sequences) and the dephosphorylated regulatory serine residues (S262,S264) are shown. The iCtr is exceptionally short, with only 29 amino acids. In accordance with its fundamental and diverse functions, it is practical to divide the iCtr into proximal and distal parts, the latter including the middle and most distal regions. Considering the final conclusions of the present study, the proximal part can also be regarded as a “gating” domain. The middle part contains two hydrophobic stretches (LIDIY and VMLFF), whereas the most distal part could be called an “interaction” domain. (The two thick horizontal lines represent the plasma membrane; EC is extracellular and IC is intracellular. The scheme is not drawn to scale). iCtr, intracellular C-terminal region; TASK, TWIK-related Acid-Sensitive K+ channel; TEVC, two-electrode voltage clamp; TMS, transmembrane segment; TRESK, TWIK-Related Spinal cord K+ channel.
	Figure 2. Analysis of phospho- and dephospho-mimicking TRESK mutants by the ENaR method. A, cartoon representation of the protein complex expressed in ENaR experiments. The ENaCα-TRESK fusion protein co-assembles with the ENaC β and γ subunits, and constitutes one TRESK and two ENaC pores. (All ENaC subunits were truncated in order to eliminate regulatory sequences). The ENaR value in a cell is defined as the ratio of K+ to Na+ current through this protein complex (in fraction form). B, recording protocol for the consecutive measurement of ENaC Na+ and TRESK K+ currents in three representative Xenopus oocytes in an ENaR experiment. In the left panel, the ENaCα-TRESK fusion construct, containing the wild-type TRESK sequence (wt), is coexpressed with ENaC β and γ. In the middle panel, ENaCα-TRESK carries the dephospho-mimicking S262A and S264A (S262,264A), whereas in the right panel, the phospho-mimicking S262E and S264E mutations (S262, 264E). ENaC and TRESK currents were measured as the Na+ current inhibited by amiloride (20 μM), and the K+ current inhibited by Ba2+ (5 mM), respectively, as indicated by the horizontal black bars. Note that the K+ current is relatively large in proportion to the Na+ current in the case of the S262, 264A mutant. C, statistical analysis of the ENaR values in three groups of oocytes expressing the constructs introduced in panel B (and indicated below the graph). Each grey circle represents the ENaR value of a single cell. The horizontal black lines with error bars indicate the average ENaR ± SD. ∗p < 0.005, one-way ANOVA, and Tukey HSD test (n = 12, 13, and 11, respectively). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio (method or value); TMS, transmembrane segment; TRESK, TWIK-Related Spinal cord K+ channel.
	Figure 3. Correlation analysis of TRESK versus ENaC currents in ENaR experiments under different experimental conditions. A, Xenopus oocytes coexpressing ENaCα-TRESK with ENaC β and γ were stimulated with ionomycin in order to activate wild-type TRESK, which had been built in the fusion construct. The amiloride-sensitive Na+ current was measured before, while the Ba2+-sensitive K+ current after the stimulation. K+ currents were plotted against Na+ currents, and each gray circle indicates a cell in the graph. Pearson’s correlation coefficient (r) is shown in the lower right corner. B, similar correlation analysis as in panel A, applying the constitutively active, S262A + S264A double mutant ENaCα-TRESK. C, In this experiment, M1 muscarinic receptor was also coexpressed with ENaCα-TRESK, ENaC β and γ. Before receptor stimulation, the correlation was weak between the small TRESK and robust ENaC currents. D, M1 receptor in the same cells as in panel C was stimulated with carbachol. TRESK was activated and the correlation improved. (Two cells did not respond to the stimulation). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio (method or value); TMS, transmembrane segment; TRESK, TWIK-Related Spinal cord K+ channel.
	Figure 4. Deletions of the iCtr decrease TRESK expression and/or channel activity. A, conventional TEVC measurement of four groups of oocytes expressing wild type (wt), Δ357, Δ366, or Δ374 TRESK, (n = 10, 6, 6, and 10), respectively. Average currents ± SD are plotted. Four-times (4×) more cRNA was microinjected in the groups of truncated TRESK channels than in the wild-type control (1×). The cells were stimulated with ionomycin (Iono., 0.5 μM), as indicated by the black bar. The Δ357 and Δ366 channels are not expressed and these curves overlap. B, ENaR analysis of the deletions in the constitutively active S264A ENaCα-TRESK (see the groups below the panel). The method of measurement and analysis was the same as in Figure 2. ∗p < 2 × 10−4, one-way ANOVA, Tukey HSD test (n = 15, 15, 15, and 14, respectively). iCtr, intracellular C-terminal region; ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 5. Conventional TEVC measurements of TRESK mutated at the K356, R361, or K367 residues. A, normalized K+ currents of four groups of oocytes expressing wild type (wt), K356A, R361A or K367A mutant TRESK, respectively. TRESK currents were measured at the ends of 300-ms voltage steps to −100 mV applied every 4 s. Extracellular [K+] was increased from 2 to 80 mM (as shown above the curves), and the oocyte was then challenged with ionomycin (Iono., 0.5 μM) as indicated by the horizontal black bar. The current measured in 2 mM EC [K+] was normalized to zero, and the current in 80 mM EC [K+], before the application of ionomycin, to one. The grey error bars represent S.D. For average current data corresponding to panels A, C, E, and H, see Fig. S6. B, Statistical analysis of TRESK activation by ionomycin from the same groups as shown in panel A (and indicated below the graph). The maximum current during the stimulation with ionomycin was divided by the resting value before the application of the ionophore (IIONO/I0), and the degree of activation was plotted for each cell as a grey circle. ∗p < 0.03, ∗∗p < 0.005, one-way ANOVA, Tukey HSD test (n = 11, 16, 12, and 12, respectively). C, the activation of the K356A + R361A double mutant TRESK by ionomycin was measured with the same protocol as in panel A, in a different cell preparation. D, statistical analysis of the activation by ionomycin in the wild type (wt) and K356A + R361A groups shown in panel C. ∗∗p < 0.005, Student’s t test (n = 9, and 6, respectively). E, normalized currents of oocytes expressing wild type (wt), or K356A + R361A TRESK, in a different cell preparation (n = 5 in both groups). Cloxyquin (CX, 100 μM) was applied before and after ionomycin (Iono., 0.5 μM) as indicated by the horizontal black bars. TRESK inhibitor A2793 (50 μM) was added after the second application of cloxyquin. F, statistical analysis of the activation by cloxyquin, using the data shown in panel E. The maximum K+ current in the presence of cloxyquin was normalized to the basal level measured before the administration of the compound (ICX/I0), and the degree of activation was plotted for each cell as a grey circle. ∗p < 0.02, Student’s t test. G, statistical analysis of the inhibition by A2793, using the data shown in panel E. The inhibition was calculated as a percentage, the inhibited current was compared to the amplitude measured before the addition of A2793 (i.e., after the second application of cloxyquin). ∗∗p < 0.005, Student’s t test. H, Normalized K+ currents of four groups of oocytes expressing wild type (wt), K356E, R361E or K356E + R361E double mutant TRESK, respectively. The measurement was similarly performed as in panel A, but in a different cell preparation. The K356E and K356E + R361E curves overlap. I, Statistical analysis of TRESK activation by ionomycin from the same groups as shown in panel H (and indicated below the graph). The method of analysis was the same as in panel B. ∗∗p < 2 × 10−4, #p = 0.076, one-way ANOVA, Tukey HSD test (n = 11, 11, 11, and 15, respectively). TEVC, two-electrode voltage clamp; TRESK, Twik-Related Spinal cord K+ channel.
	Figure 6. ENaR analysis of the K356E and R361E mutations in the wild type, constitutively active S262,264A, and inhibited S262,264D ENaCα-TRESK channels. A, ENaR values are plotted from six groups of oocytes expressing ENaCα-TRESK with or without the S262A + S264A double mutation, and containing wild type, K356E, or R361E mutant versions of the iCtr, as indicated below the graph. The method of measurement (coexpression of truncated ENaC β and γ, amiloride- and Ba2+-sensitive currents, etc.) and analysis was the same as introduced in Figure 2. ∗p < 2 × 10−4, ns: not significant, one-way ANOVA, Tukey HSD test (n = 13, 13, 13, 13, 12 and 7, respectively). B, comparison of the ENaR values of the phospho-mimicking S262D + S264D and the dephospho-mimicking S262A + S264A double mutants of ENaCα-TRESK. Data are repeated in Fig. S1C. ∗∗p < 10−6, Student’s t test (n = 12, and 16, respectively). C, the missing effect of the S262D + S264D versus S262A + S264A mutations on the ENaR values in the K356E and R361 mutant ENaCα-TRESK channels. Groups are indicated below the graph. Note the different vertical scales in panels B and C ns: not significant, one-way ANOVA, Tukey HSD test (n = 8, 8, 12, and 6, respectively). (Panels A, B, and C correspond to three different cell preparations, respectively). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 7. Elimination of the hydrophobic modules of the distal iCtr and the F372L mutation reduces the ENaR of the constitutively active S264A ENaCα-TRESK. ENaR values are plotted from five groups of oocytes expressing wild type (wt, negative control), S264A (positive control), or three other modified versions of S264A ENaCα-TRESK, containing the replacement of the RLIDIY sequence with RAADAA, the substitution of VMLFF sequence with five alanines (VMLFF-5A), or the F372L mutation, respectively, as indicated below the graph. The method of measurement was the same as in Figure 2. ∗p < 0.005, ∗∗p < 2 × 10−4, one-way ANOVA, Tukey HSD test (n = 12, 23, 11, 11, and 11, respectively). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 8. Diverse modifications of the most distal iCtr are compatible with high TRESK activity. The most distal iCtr of S262A + S264A double mutant ENaCα-TRESK was replaced with 12 lysines (12K), asparagines (12N), aspartates (12D), or a shorter combination of lysines and tryptophans (4K4W2K). ENaR values of these constructs, and the positive control S262A + S264A channel, were plotted, as indicated below the graph. The method of measurement was the same as in Figure 2. ∗p < 2 × 10−4, one-way ANOVA, Tukey HSD test (n = 13, 20, 12, 9, and 12, respectively). ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region.
	Figure 9. Effects of the fusion of K-Ras 4B membrane-anchoring motif with TRESK iCtr on the K+ currents and ENaR values, before and after ionomycin. A, conventional TEVC measurement of four groups of oocytes expressing wild type (wt), 356-6Kcaax, 367-6Kcaax, or 375-6Kcaax TRESK, (n = 10, 6, 6, and 12), respectively. Average currents ± S.D. are plotted. The cells were stimulated with ionomycin (Iono., 0.5 μM), as indicated by the black bar. The 356-6Kcaax and 367-6Kcaax channels are not expressed and these curves overlap. B, ENaR analysis of the 6Kcaax modifications in ENaCα-TRESK (see the groups below the panel). The method of measurement was the same as in Figure 2; however, the Ba2+-sensitive K+ current was measured twice in each cell, before (Iono. −) and after the application of ionomycin (Iono. +, 0.5 μM, as indicated below the graph). ∗p < 2 × 10−4, #p = 0.068, ns: not significant, two-way mixed-design ANOVA, Tukey HSD test (n = 15, 14, and 14, respectively). (The 375-6Kcaax Iono + group is different from all the other groups at p < 2 × 10−4, whereas the effect of ionomycin in the wild-type group was not significant in this experiment). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 10. Confocal micrograph showing the localization of EGFP fused to TRESK iCtr. Confocal microscope images of HEK293A cells expressing the enhanced green fluorescent protein (EGFP, left panel), or the EGFP-hTRESK-iCtr construct (right panel). The addition of the 29 amino acids of TRESK iCtr to the C-terminus of EGFP prevents uniform cytoplasmic localization. The magnification is the same on both panels. For a more extensive set of images, see Fig. S7. EGFP, enhanced green fluorescent protein; ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 11. RW361 modification of TRESK iCtr results in extraordinarily high ENaR values and decreases channel expression. A, conventional TEVC measurement of four groups of oocytes expressing wild type (wt), S264A, RW361, or S264A + RW361 TRESK constructs (n = 12, 11, 5, and 12, respectively). Protocol was the same as in Figure 4A. Only small K+ currents were induced by the expression of RW361 and S264A + RW361 constructs; these curves partially overlap. B, Representative current recording from an oocyte microinjected with eight times (8×) higher amount of cRNA of the RW361 construct than in panel A. This cell evidently expressed TRESK current, which was activated in response to ionomycin (Iono., 0.5 μM, black bar), although the degree of activation could not be calculated because the K+ current was in the range of endogenous K+ conductance of the oocyte (see the vertical scale bar). C, ENaR analysis of the wild type (wt), S264A, RW361, and S264A + RW361 ENaCα-TRESK constructs, as indicated below the graph. The method of measurement was the same as in Figure 2. The reorganization of the distal iCtr by the RW361 modification resulted in remarkably high ENaR values in the RW361 and S264A + RW361 groups (note the vertical scale). ∗p < 2 × 10−4, ns: not significant, one-way ANOVA after logarithmic transformation, Tukey HSD test (n = 12, 23, 11, and 15, respectively). ENaC, epithelial Na+ channel; ENaR, epithelial sodium current ratio; iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel, K2P18.1, KCNK18.
	Figure 12. Single-channel analysis of S264A mutant TRESK channels, truncated at amino acid 374, or modified by the replacement of the iCtr with RW361 sequence. A, representative single channel current recordings from two Xenopus oocyte membrane patches in the cell-attached configuration. In the upper recording, the patch contained S264A + Δ374 TRESK channels (more than one), while in the lower recording, a single S264A + RW361 TRESK channel was present. Zero current levels are indicated with horizontal arrows. The potential of the pipette solution was clamped to −100 mV. For more recordings, see Fig. S7. B, statistical analysis of the NPo values of S264A + Δ374 and S264A + RW361 TRESK channels, as indicated below the graph. The grey circles represent the average NPo from different membrane patches. ∗p < 0.01, Student’s t test (n = 5, and 6, respectively). C, statistical analysis of the burst duration of S264A + Δ374 and S264A + RW361 TRESK channels, as indicated below the graph. The gray circles represent the average burst duration from different membrane patches. ∗p < 0.03, Student’s t test (n = 5 in both groups). iCtr, intracellular C-terminal region; TRESK, Twik-Related Spinal cord K+ channel.

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