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- W2014096411 abstract "The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3η with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3η accelerated the return of the K+ current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3η was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The γ and η isoforms of 14-3-3 controlled TRESK regulation, whereas the β, ζ, ∈, σ, and τ isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3η. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K+ channel family. The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3η with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3η accelerated the return of the K+ current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3η was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The γ and η isoforms of 14-3-3 controlled TRESK regulation, whereas the β, ζ, ∈, σ, and τ isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3η. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K+ channel family. Members of the 14-3-3 family are dimeric proteins, and each subunit possesses a single polypeptide binding groove (1Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). Proteins that bind these grooves typically encode either the RSXpSXP (mode I) or RX(Y/F)XpSXP (mode II) consensus motif (where X may be any amino acid, and pS denotes phosphoserine (2Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 3Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Several different interacting partners of 14-3-3 have been described, and 14-3-3 proved to be an important constituent of large protein complexes implicated in such diverse processes as the initiation of DNA replication, transcription, control of cell cycle, intracellular trafficking, and the modulation of ion channel function (4Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (627) Google Scholar, 5Gardino A.K. Smerdon S.J. Yaffe M.B. Semin. Cancer Biol. 2006; 16: 173-182Crossref PubMed Scopus (214) Google Scholar).Two-pore domain potassium (2PK+) channels give rise to background (leak) K+ currents that are pivotal regulators of the excitability in neurons and other cell types (6Lotshaw D.P. Cell Biochem. Biophys. 2007; 47: 209-256Crossref PubMed Scopus (139) Google Scholar). Members of this potassium channel family attracted particular attention as the stimulation of their currents essentially contributed to the therapeutically important action of volatile anesthetics (7Patel A.J. Honore E. Lesage F. Fink M. Romey G. Lazdunski M. Nat. Neurosci. 1999; 2: 422-426Crossref PubMed Scopus (565) Google Scholar, 8Heurteaux C. Guy N. Laigle C. Blondeau N. Duprat F. Mazzuca M. Lang-Lazdunski L. Widmann C. Zanzouri M. Romey G. Lazdunski M. EMBO J. 2004; 23: 2684-2695Crossref PubMed Scopus (422) Google Scholar, 9Liu C.H. Au J.D. Zou H.L. Cotten J.F. Yost C.S. Anesth. Analg. 2004; 99: 1715-1722Crossref PubMed Scopus (92) Google Scholar, 10Keshavaprasad B. Liu C. Au J.D. Kindler C.H. Cotten J.F. Yost C.S. Anesth. Analg. 2005; 101: 1042-1049Crossref PubMed Scopus (41) Google Scholar). Among the 15 2PK+ channels, so far only TASK-1 and TASK-3 subunits have been shown to interact with 14-3-3β and -ζ through an unconventional (mode III) C-terminal motif. The binding of 14-3-3 overrides the endoplasmic reticulum retention signal and redirects these TASK channels to the cell surface (11O'Kelly I. Butler M.H. Zilberberg N. Goldstein S.A.N. Cell. 2002; 111: 577-588Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 12Rajan S. Preisig-Muller R. Wischmeyer E. Nehring R. Hanley P.J. Renigunta V. Musset B. Schlichthorl G. Derst C. Karschin A. Daut J. J. Physiol. (Lond.). 2002; 545: 13-26Crossref Scopus (123) Google Scholar, 13Renigunta V. Yuan H. Zuzarte M. Rinne S. Koch A. Wischmeyer E. Schlichthorl G. Gao Y. Karschin A. Jacob R. Schwappach B. Daut J. Preisig-Muller R. Traffic. 2006; 7: 168-181Crossref PubMed Scopus (83) Google Scholar).TRESK, 2The abbreviations used are: TRESK, TWIK-related spinal cord K+ channel; PKA, protein kinase A; NFAT, nuclear factor of activated T cells; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; Ni-NTA, nickelnitrilotriacetic acid. 2The abbreviations used are: TRESK, TWIK-related spinal cord K+ channel; PKA, protein kinase A; NFAT, nuclear factor of activated T cells; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; Ni-NTA, nickelnitrilotriacetic acid. the 15th member of the 2PK+ channel family, was cloned from human spinal cord (14Sano Y. Inamura K. Miyake A. Mochizuki S. Kitada C. Yokoi H. Nozawa K. Okada H. Matsushime H. Furuichi K. J. Biol. Chem. 2003; 278: 27406-27412Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and mouse cerebellum (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Its mRNA is also expressed in the testis (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), spleen, thymus, placenta (16Kang D. Mariash E. Kim D. J. Biol. Chem. 2004; 279: 28063-28070Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and in the cerebrum (9Liu C.H. Au J.D. Zou H.L. Cotten J.F. Yost C.S. Anesth. Analg. 2004; 99: 1715-1722Crossref PubMed Scopus (92) Google Scholar). Recently, TRESK current was detected with painstaking work at the single channel level in dorsal root ganglion neurons (17Kang D. Kim D. Am. J. Physiol. Cell Physiol. 2006; 291: 138-146Crossref PubMed Scopus (167) Google Scholar), and others suggested that it was responsible for about 20% of the background K+ current in these cells (18Dobler T.M. Springauf A. Tovornik S. Weber M. Schmitt A. Sedlmeier R. Wischmeyer E. Döring F. J. Physiol. (Lond.). 2007; 585: 867-879Crossref Scopus (116) Google Scholar). However, macroscopic TRESK current still could not be reliably separated from the current of other 2PK+ channels in the absence of specific inhibitors.We have recently demonstrated that TRESK is regulated in a unique manner. The channel, expressed heterologously in Xenopus oocytes, is reversibly activated by calcium via the calcium/calmodulin-dependent protein phosphatase, calcineurin. Serine 276 was identified as the likely target of calcineurin-mediated dephosphorylation in the channel (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In addition to its enzymatic action, calcineurin is also recruited to a nuclear factor of activated T cells (NFAT)-like binding motif (PQIVID) of TRESK (19Czirják G. Enyedi P. J. Biol. Chem. 2006; 281: 14677-14682Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In the present study we demonstrate that 14-3-3 proteins also bind directly to the intracellular loop of TRESK and control the calcium-dependent regulation of the channel.EXPERIMENTAL PROCEDURESPlasmids—The sequences of pXEN and pXEN-pQ108 vectors were deposited to GenBank™ under the accession numbers EU267939 and EU267940, respectively. pXEN-pQ108, coding the flexible, artificial polypeptide chain (LEHQQQQQQQQQ)9, was obtained by inserting nine orientationally ligated pQ-s/pQ-a 5′-phosphorylated primer dimers into the Eco130I site of pXEN (see supplemental Table 1 for primer sequences). The (LEHQ9)9 polypeptide (approximating a random coil of 108 amino acids with a predicted contour length of about 41 nm) was designed on the basis of the known structure of polyglutamine peptides (20Masino L. Kelly G. Leonard K. Trottier Y. Pastore A. FEBS Lett. 2002; 513: 267-272Crossref PubMed Scopus (134) Google Scholar, 21Sen S. Dash D. Pasha S. Brahmachari S.K. Protein Sci. 2003; 12: 953-962Crossref PubMed Scopus (30) Google Scholar) and the N-terminal inactivation chain of Drosophila Shaker K+ channel (22Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1270) Google Scholar). The coding regions of mouse TRESK and human 14-3-3η (the latter differs only in two amino acids from the mouse ortholog) were amplified by PCR from our pEXO-mTRESK clone (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and pcDNA3.1-h14-3-3η (received from Andrey S. Shaw) with the mTRESK-s/mTRESK-a and h14-3-3η-s/h14-3-3η-a primer pairs, respectively. The 14-3-3 and TRESK products were double-digested with PaeI/XhoI and EcoRI/Kpn2I, respectively, and cloned between the corresponding sites of pXEN-pQ108, resulting in the pXEN-mTRESK-pQ108-h14-3-3η (“chained”) construct.Wild type and R57A,R61A mutant human 14-3-3η were subcloned from the mammalian expression vectors to pXEN with EcoRI/XhoI. The other six 14-3-3 isoforms were amplified with Pfu polymerase (Fermentas, Vilnius, Lithuania) from mouse brain RNA after reverse transcription, applying the m14-3-3x-s/m14-3-3x-a primer pairs (where x stands for β, γ, ∈, ζ, σ and τ, respectively). The PCR protocol was 2 min initial denaturation at 98 °C, 34 cycles of 30-s denaturation at 98 °C, 1 min annealing, 80–90-s extension at 72 °C, and 5 min final extension at 72 °C; the annealing temperature was 64, 61, 59, and 56 °C in the first 3, second 3, third 3, and final 25 cycles, respectively. The products were digested at the respective restriction enzyme sites (see supplemental Table 1) and ligated to EcoRI/XhoI double-digested pXEN. The dominant negative versions of these isoforms (R58A,R62A β; R57A,R61A γ; R57A,R61A ∈; R56A,R60A ζ; R56A,R60A σ, and RA56,R60A τ) were produced with QuikChange site-directed mutagenesis (see the sequences of the sense primers in supplemental Table 1). The resulting clones were verified by automatic sequencing.The construction of the GST-TRESKloop-TAPtag fusion protein was described previously (19Czirják G. Enyedi P. J. Biol. Chem. 2006; 281: 14677-14682Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The GST-h14-3-3η constructs were obtained by subcloning the EcoRI/XhoI inserts of wild type and dominant negative h14-3-3η (see above) to pGEX-6P-3 (Amersham Biosciences). The thioredoxin-hexahistidine-h14-3-3η (Trx-His6-h14-3-3η) plasmids were produced by ligating the above inserts to the EcoRI/XhoI double-digested pET32-ΔKpn vector (pET32-ΔKpn derived from pET32a+ (Novagen, Madison, WI) by digesting the plasmid with KpnI, polishing its sticky ends with Klenow polymerase, and religating). To produce the TRESKloop-H8 construct, the DNA coding for amino acids 185–292 of mouse TRESK was amplified by two consecutive rounds of PCR with the mTRloop-s/mTRloop-H8-a1 and mTRloop-s/mTRloop-H8-a2 primer combinations. The PCR product (coding also the C-terminal eight histidines) was double-digested with NcoI/XhoI and ligated to pET15b (Novagen). The 10 serines and 1 threonine of TRESKloop-H8 were sequentially mutated to alanine in different combinations by QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) to obtain the proteins containing only 1, 2, or 3 serines in the desired patterns (see the sense primers used for the mutations in supplemental Table 1).Animals, Tissue Preparation, Xenopus Oocyte Microinjection, and Two-electrode Voltage Clamp Measurements—The tissues for RNA preparation derived from NMRI mouse strain (Toxicop). The oocytes were prepared, the cRNA was synthesized and microinjected, and two-electrode voltage clamp measurements were performed as previously described (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 23Czirják G. Enyedi P. J. Biol. Chem. 2002; 277: 5426-5432Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Oocytes were injected 1 day after defolliculation. Fifty nanoliters of the appropriate RNA solution was delivered with the Nanoliter Injector (World Precision Instruments, Sarasota, FL). Electrophysiological experiments were performed 3 or 4 days after the injection. Low [K+] solution contained 95 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 5 mm HEPES (pH 7.5 adjusted with NaOH). High [K+] solution contained 80 mm K+ (78 mm Na+ of the low [K+] solution was replaced with K+). All treatments of the animals were conducted in accordance with state laws and institutional regulations. The experiments were approved by the Animal Care and Ethics Committee of Semmelweis University.Production and Purification of Recombinant Proteins—The GST fusion constructs were expressed in the BL21 strain of Escherichia coli. Bacteria were sonicated in G-lysis solution containing 50 mm Tris-HCl (pH 7.6), 50 mm KCl, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 1 mm PMSF, 0.1 mm benzamidine. The lysate was centrifuged, and the GST fusion proteins were affinity-purified from the supernatant with glutathione-agarose (Sigma). In the GST pulldown experiments, these immobilized proteins were used. For other experiments, the proteins were eluted from the resin with G-lysis supplemented with 20 mm glutathione and dialyzed against a solution containing 10 mm HEPES, 100 mm NaCl, 50 mm KCl, 2 mm MgCl2, 1 mm β-mercaptoethanol (pH 7.4 with NaOH).The bacteria expressing the thioredoxin-His-tag fusion proteins were lysed in N-lysis solution containing 30 mm phosphate, 200 mm NaCl, 2 mmMgCl2, 1 mm PMSF, 0.2 mm benzamidine, 5 mm imidazole, 5.6 mm β-mercaptoethanol (pH 7.8 with NaOH). The proteins were affinity-purified with Ni-NTA agarose (Qiagen, Chatsworth, CA). The resin was washed 6 times for 5 min with 12 ml of N-lysis solution (in the second and third pairs of washing steps, the imidazole concentration of N-lysis solution was increased to 20 and 50 mm, respectively). The proteins were eluted with N-lysis containing 256 mm imidazole and dialyzed against a solution containing 20 mm Tris-HCl (pH 7.3), 100 mm NaCl, 50 mm KCl, 2 mm MgCl2, 1 mm EGTA, and 0.1 mm dithiothreitol.TRESKloop-H8 proteins (wild type and multiple mutants) accumulated in inclusion bodies; therefore, these proteins were purified under denaturing conditions. The inclusion bodies were dissolved at room temperature in IB-lysis solution containing 30 mm phosphate, 200 mm NaCl, 1 mm PMSF, 0.1 mm benzamidine, 5 mm β-mercaptoethanol, 5 mm imidazole, 7000 mm urea (pH 7.8 with NaOH). TRESKloop-H8 proteins were purified with Ni-NTA-agarose from this solution. The resin was washed (at room temperature) 3 times with IB-lysis solution of increased (50 mm) imidazole concentration, once with IB-lysis, twice with IB-lysis of pH 5.5, and once again with IB-lysis. TRESKloop-H8 proteins immobilized on Ni-NTA resin were stored as a 50% suspension in IB-lysis solution at 4 °C.In Vitro Radioactive Phosphorylation—For the preparation of Xenopus oocyte cytosol, ≈1 g of ovarian lobes was homogenized in 1 ml of a solution containing 50 mm HEPES, 50 mm KCl, 10 mm MgCl2, 50 mm β-glycerol phosphate, 20 mm NaF, 20 mm para-nitrophenylphosphate, 0.2 mm sodium orthovanadate, 2 mm PMSF, 0.2 mm benzamidine, 2 mm β-mercaptoethanol, 10 mm imidazole supplemented with cyclosporine A (5 μm), FK506 (1 μm), leupeptin (0.5 mg/ml), aprotinin (0.5 mg/ml), and soybean trypsin inhibitor (1 mg/ml, Sigma, type IIS), pH adjusted to 7.2 with NaOH. The lysate was centrifuged 3 times at 16,000 × g for 10 min and always the middle phase (supernatant above the insoluble pellet and below the lipid layer at the surface) was taken to the next centrifugation. The cleared supernatant was incubated with Ni-NTA-agarose (200 μl) to deplete the proteins binding nonspecifically to this resin.TRESKloop-H8 proteins immobilized on 12–25 μl of Ni-NTA resin were washed 3 times with EQ solution containing 50 mm HEPES, 50 mm KCl, 1 mm PMSF, 0.1 mm benzamidine, 2 mm β-mercaptoethanol (pH 7.2 with NaOH) 20 min before the phosphorylation reaction. The immobilized proteins were phosphorylated at room temperature for 40 min in the presence of 100 μl of Xenopus oocyte cytosol and 1 MBq [γ-32P]ATP with continuous shaking. The proteins were run on 15% SDS-PAGE gels, the gels were stained with Coomassie Brilliant Blue, and their radioactivity was detected with phosphorimaging (GS-525, Bio-Rad). The phosphorylation with protein kinase A holoenzyme (protein kinase A (PKA), 1 μg/reaction, Sigma P5511, 0.7 units/μg) was performed at 37 °C for 30 min in a solution containing 20 mm HEPES, 80 mm KCl, 10 mm MgCl2, 25 mm β-glycerol phosphate, 0.5 mm β-mercaptoethanol, 0.1 mm sodium orthovanadate, 1 mm cAMP (pH 7.5 with NaOH) supplemented with 50 μm Na2ATP and 50 kBq [γ-32P]ATP.GST and His-tag Pulldown Assays—The immobilized GST fusion proteins were phosphorylated overnight with PKA at 37 °C in a solution containing 50 mm Tris-HCl (pH 7.5), 50 mm KCl, 10 mm MgCl2, 50 mm β-glycerol phosphate, 1.3 mm dithiothreitol, 0.2 mm sodium orthovanadate, 1 mm cAMP, and 5 mm Na2ATP supplemented with 1% Triton X-100. The resins were washed twice with 0.2 ml G-binding solution containing 50 mm Tris-HCl (pH 7.5), 50 mm NaCl, 50 mm KCl, 1 mm PMSF, 0.1 mm benzamidine, 1 mm EDTA, 1 mm dithiothreitol. In the GST pulldown assay (with the Trx-His6-h14-3-3η proteins) the 1-h binding reaction was performed at 4 °C by rotating the beads in G binding solution containing 1% Triton X-100. The unbound proteins were removed by two washing steps (each 1 min) with 1 ml of G binding solution (+1% Triton X-100 in the first step).For His-tag pulldown assays, the different immobilized TRESKloop-H8 proteins were washed 3 times with H-phos solution containing 50 mm HEPES, 50 mm KCl, 10 mm MgCl2, 50 mm β-glycerol phosphate, 2 β-mercaptoethanol, and 1% Triton X-100 (pH 7.5 with NaOH). The proteins were phosphorylated overnight with PKA at 37 °C in H-phos solution supplemented with 20 mm imidazole, 5 mm Na2ATP, 1 mm cAMP, and 200 μm sodium orthovanadate (pH 7.5 with NaOH). The His-tag pulldown assays (of the GST-h14-3-3η proteins) were performed in H-binding solution containing 50 mm HEPES, 50 mm NaCl, 50 mm KCl, 1 mm PMSF, 0.1 mm benzamidine, 2 mm β-mercaptoethanol, 30 mm imidazole (pH 7.4 with NaOH).RESULTS14-3-3 Proteins Determine the Recovery of TRESK Current from the Calcium-dependent Activation—The intracellular loop of mouse TRESK contains two putative 14-3-3 binding sites (KWRSLP194 and RSNSCP266, identified with Motif Scan). Assuming that 14-3-3 is really anchored to either of these sites in TRESK, it is reasonable to expect that 14-3-3 is also involved in a function related to the background K+ channel. Therefore, we examined whether the experimental manipulation of the cellular level of 14-3-3 proteins modified the functional properties of TRESK. Because the coexpression of 14-3-3η with mouse or human TRESK did not influence the resting TRESK current (data not shown), we tested whether 14-3-3 modulated the calcium-dependent regulation of the channel.We have previously reported (15Czirják G. Tóth Z.E. Enyedi P. J. Biol. Chem. 2004; 279: 18550-18558Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) that the calcium ionophore, ionomycin, induced a large (5–15-fold) activation of TRESK current expressed in Xenopus oocytes. The calcium-dependent activation was reversible after the cessation of the calcium signal, but the return of the background K+ current to its resting state (decrease in the current, hereafter called “recovery,” Fig. 1A) varied widely among the different oocyte preparations (e.g. the recovery was 20 ± 3% (n = 6) after 5 min in one oocyte preparation, whereas it was 73 ± 6% (n = 8) in another). These highly different kinetics of recovery may have reflected different concentrations of endogenous 14-3-3. To detect the effect of 14-3-3 irrespective of the endogenous expression, we experimentally manipulated the functional 14-3-3 levels in both direction for each oocyte preparation. In one of the compared groups of oocytes, the concentration of 14-3-3 was increased by the coexpression of the human 14-3-3η with TRESK. In the other group, the functional 14-3-3 level was reduced by the coexpression of the dominant negative R57A,R61A mutant form of human 14-3-3η with the K+ channel (the literature frequently refers to the dominant negative mutant as R56A,R60A 14-3-3η, although this numbering does not apply to the η isoform). To illustrate the effect of endogenous 14-3-3, we also show the results from oocytes expressing only TRESK (gray curves in Fig. 1).In addition to the conventional normalization of the data to the resting current (“normalized current,” see the legend to Fig. 1A), the same recordings were also presented in another form; they were normalized to the value measured at the end of ionomycin stimulation (normalized recovery, also defined in the legend to Fig. 1A). The latter normalization shows to what degree the activated current returned to the resting level in a given time after the withdrawal of ionomycin.The coexpression of the wild type or the dominant negative 14-3-3 with mouse TRESK had significantly different effects on the activation of the K+ current in one oocyte preparation. The activation was 9.4 ± 0.7-fold (n = 5) at the end of the application of ionomycin in the cells coexpressing the wild type 14-3-3, whereas it was 19.1 ± 2.8-fold (n = 5) in the cells coexpressing the dominant negative 14-3-3 with mouse TRESK (p < 0.02 at 276s(t test), Fig. 1B), indicating that 14-3-3 inhibited the activation of mouse TRESK. However, in another oocyte preparation no significant change in mouse TRESK activation was observed (Fig. 1D). As was also verified by the microinjection of 14-3-3 protein or the immobilization of 14-3-3 to the channel (see below), the activation of mouse TRESK was undoubtedly suppressed by high concentration of 14-3-3. Perhaps this inhibitory effect was not apparent in every coexpression experiment, because it also depended on the level of overexpression of 14-3-3. The activation of human TRESK was not modified by the coexpression of the wild type or the dominant negative 14-3-3η with the channel (Fig. 1F).The recovery of mouse TRESK (see the end of the recordings in Fig. 1, C and E) was reduced to 5 ± 6 and 15 ± 6% in the first and second oocyte preparation (n = 5 and 6), respectively, by the coexpression of wild type 14-3-3η with the channel. In the cells coexpressing the dominant negative 14-3-3 with mouse TRESK, the recovery was 46 ± 1 and 45 ± 4% (n = 5, p < 10–4 and n = 6, p < 0.003) in the two preparations, respectively (Fig. 1, C and E). In general, care must be taken when evaluating the recovery if the activation is also different in the compared groups (as in the first oocyte preparation, see Fig. 1, B and C). However, in this case the rate of recovery in the cells coexpressing the dominant negative 14-3-3 was substantially higher than that in the cells coexpressing the wild type 14-3-3. At the end of the measurement, neither the currents (11.6 ± 1.9 μAinthe wild type and 10.0 ± 1.6 μA in the dominant negative group) nor the normalized currents (9.3 ± 0.8- and 11.0 ± 1.8-fold, respectively, at 591 s in Fig. 1B) were significantly different. Despite this, the rate of recovery (estimated by the slope of the normalized currents at the end of the measurement in Fig. 1B) was about 5-fold higher in the dominant negative than in the wild type group (3.9 ± 1.1 versus 0.8 ± 0.2% of the resting current per second, p < 0.03). Because TRESK expression was nearly equal in the different oocytes in this experiment, the rates calculated even from the original current recordings were significantly different at the end of the measurement (26.3 ± 9.0 and 5.7 ± 3.1 nA/s, p < 0.05, respectively). This clearly indicates that 14-3-3 inhibits the recovery of mouse TRESK in addition to its effect on the activation of the channel. This result was also confirmed by the data obtained in the second oocyte preparation, where the activation was not significantly influenced (Fig. 1D), but the recovery was robustly inhibited by 14-3-3 (Fig. 1E).Although the degree of activation of human TRESK was not affected by 14-3-3, its recovery was inhibited. The recovery was 12 ± 4% in the cells coexpressing the wild type (n = 7), whereas it was 52 ± 6% in the group coexpressing the dominant negative 14-3-3η with the channel (n = 7, p < 0.002 at 483 s, Fig. 1G).In summary, 14-3-3η suppressed the calcium-dependent activation of mouse TRESK (this effect was not significant in all of the examined oocyte preparations). However, the major effect of 14-3-3 was statistically significant in all of our experiments; 14-3-3η inhibited the recovery of both human and mouse TRESK currents.The Effect of 14-3-3η Is Acute and Spatially Restricted to TRESK in the Cell—14-3-3 proteins are known to affect a plethora of cellular functions including the regulation of the cell cycle and gene expression. In the above experiments, a high amount of coexpressed 14-3-3η was present in the oocytes for days (see supplemental Fig. S1). To exclude that gene expression changes or other long term alterations were responsible for the effects of 14-3-3η on TRESK regulation, we examined whether the microinjection of a recombinant 14-3-3 protein also inhibited TRESK recovery similarly to the coexpression of 14-3-3.In this experiment R57A,R61A 14-3-3η was coexpressed with TRESK to reduce the effect of endogenous 14-3-3 proteins. Before the ionomycin application, the cells were microinjected either with wild type GST-14-3-3η or (as a control) with R57A,R61A mutant GST-14-3-3η protein. The activation of mouse TRESK was smaller in the wild type (10.1 ± 1.3-fold, n = 6) than in the R57A,R61A group (19.5 ± 1.2-fold activation (n = 5), p < 0.001 at 213 s, Fig. 2A). The microinjection of GST-14-3-3η also inhibited the return of TRESK current to the resting state (1 ± 10% recovery in the GST-14-3-3η-injected and 41 ± 2% in the GST-14-3-3η-R57A,R61A-injected cells, p < 0.01 at 429s, Fig. 2B). The rate of recovery was also significantly different at the end of the measurement in the two groups (2.8 ± 0.3 and 5.1 ± 0.4% of the resting current per second, respectively, p < 0.01, Fig. 2A; at this time the currents and the normalized currents were not different in the two groups (data not shown)). The same microinjection experiment was also performed with oocytes expressing only mouse TRESK, and the activation and the recovery were also significantly attenuated by the microinjection of wild type GST-14-3-3η in these cells (results not shown).FIGURE 2The microinjection of recombinant GST-h14-3-3η protein inhibits the activation of mouse TRESK and delays the recovery from activation of both human and mouse TRESK channels. A, normalized currents of two groups of oocytes microinjected with 50 nl of GST-h14-3-3η (wild type, wt., black curve) or R57A,R61A mutant GST-h14-3-3η fusion protein (gray curve). The cells were coexpressing mouse TRESK with R57A,R61A 14-3-3η (in both groups). Extracellular [K+] was increased from 2 to 80 mm, and the oocytes were challenged with ionomycin (Iono., 0.5 μm, as indicated by the horizontal black bar) 108–248 min after the microinjection of the proteins. B, normalized recovery was calculated from the same recordings as represented in panel A. C and D, a similar microinjection experiment as in A and B was performed with human TRESK in another oocyte preparation. The proteins were microinjected 188–236 min before the application of ionomycin.View Large Image Figure ViewerDownload Hi-res i" @default.
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- W2014096411 title "Phosphorylation-dependent Binding of 14-3-3 Proteins Controls TRESK Regulation" @default.
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