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• W2090145908 abstract "TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca2+. In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl-, as demonstrated in ion substitution experiments using the cation N-methyl-d-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl- with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels. TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca2+. In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl-, as demonstrated in ion substitution experiments using the cation N-methyl-d-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl- with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels. The members of the transient receptor potential (TRP) 3The abbreviations used are: TRPtransient receptor potentialMES4-morpholineethanesulfonic acidDIDS4,4′-diisothiocyanostilbene-2,2′-disulfonic acidNMDGN-methyl-d-glucamineADPRADP-ribose. 3The abbreviations used are: TRPtransient receptor potentialMES4-morpholineethanesulfonic acidDIDS4,4′-diisothiocyanostilbene-2,2′-disulfonic acidNMDGN-methyl-d-glucamineADPRADP-ribose. family of non-selective cation channels display an extraordinarily broad spectrum of activation mechanisms reflecting their involvement in manifold biological processes. The basic architecture of TRP channels is shared with the well characterized voltage-gated K+ channels. Here, the transmembrane segment S6 contains the activation gate that opens the pore in response to the movement of the activated S4 voltage sensor (1Long S.B. Campbell E.B. McKinnon R. Science. 2005; 309: 903-908Crossref PubMed Scopus (809) Google Scholar, 2Lu Z. Klem A.M. Ramu Y. J. Gen. Physiol. 2005; 120: 663-676Crossref Scopus (258) Google Scholar). Although TRP channels do not contain a classical voltage sensor, one may presume an interaction of S6 with the pore that governs functional relevance for the properties of TRP channels.TRPM2 and TRPM8 of the melastatin-related subfamily of TRP channels are the closest relatives within the TRP family (42% identical residues, Ref. 3Peier A.M. Moqrich A. Hergarden A.C. Reeve A.J. Andersson D.A. Story G.M. Earley T.J. Dragoni I. McIntyre P. Bevan S. Patapoutian A. Cell. 2002; 108: 705-715Abstract Full Text Full Text PDF PubMed Scopus (1695) Google Scholar) but their activation mechanisms are completely different. The principal activators of TRPM2 are intracellular ADP-ribose (ADPR) and reactive oxygen species such as hydrogen peroxide (4Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (736) Google Scholar, 5Wehage E. Eisfeld J. Heiner I. Jüngling E. Zitt C. Lückhoff A. J. Biol. Chem. 2002; 277: 23150-23156Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Accordingly, TRPM2 is involved in the cellular responses to oxidative and nitrative stress (6Hara Y. Wakamori M. Ishii M. Maeno E. Nishida M. Yoshida T. Yamada H. Shimizu S. Mori E. Kudoh J. Shimizu N. Kurose H. Okada Y. Imoto K. Mori Y. Mol. Cell. 2002; 9: 163-173Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 7Zhang W. Chu X. Tong Q. Cheung J.Y. Conrad K. Masker K. Miller B.A. J. Biol. Chem. 2003; 278: 16222-16229Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 8Zhang W. Hirschler-Laszkiewicz I. Tong Q. Contrad K. Sun S.C. Penn L. Barber D.L. Stahl R. Carey D.J. Cheung J.Y. Miller B.A. Am. J. Physiol. 2006; 290: C1146Crossref PubMed Scopus (105) Google Scholar, 9Kraft R. Grimm C. Grosse K. Hoffmann A. Sauerbruch S. Kettenmann H. Schultz G. Harteneck C. Am. J. Physiol. 2004; 286: 129-137Crossref PubMed Scopus (222) Google Scholar, 10Fonfria E. Marshall I.C. Benham C.D. Boyfield I. Brown J.D. Hill K. Hughes J.P. Skaper S.D. McNulty S. Br. J. Pharmacol. 2004; 143: 186-192Crossref PubMed Scopus (227) Google Scholar, 11Fonfria E. Marshall I.C. Boyfield I. Skaper S.D. Hughes J.P. Owen D.E. Zhang W. Miller B.A. Benham C.D. McNulty S. J. Neurochem. 2005; 95: 715-723Crossref PubMed Scopus (160) Google Scholar, 12Perraud A.L. Takanishi C.L. Shen B. Kang S. Smith M.K. Schmitz C. Knowles H.M. Ferraris D. Li W. Zhang J. Stoddard B.L. Scharenberg A.M. J. Biol. Chem. 2005; 280: 6138-6148Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Kaneko S. Kawakami S. Hara Y. Wakamori M. Itoh E. Minami T. Takada Y. Kume T. Katsuk H. Mori Y. Akaike A. J. Pharmacol. Sci. 2006; 101: 66-76Crossref PubMed Scopus (170) Google Scholar, 14Grubisha O. Rafty A.L. Takanishi C.L. Xu X. Tong L. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2006; 281: 14057-14065Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).The TRPM8 channel mediates the cold sensation of the somatosensory system (3Peier A.M. Moqrich A. Hergarden A.C. Reeve A.J. Andersson D.A. Story G.M. Earley T.J. Dragoni I. McIntyre P. Bevan S. Patapoutian A. Cell. 2002; 108: 705-715Abstract Full Text Full Text PDF PubMed Scopus (1695) Google Scholar, 15Reid G. Babes A. Pluteanu F. J. Physiol. 2002; 542: 595-614Crossref Scopus (180) Google Scholar, 16McKemy D.D. Neuhausser W.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (1938) Google Scholar) but was initially discovered as a protein with up-regulated expression in prostate cancer and some other malignant tissues; these roles of TRPM8 are apparently independent of any significant temperature variations (17Tsavaler L. Shapero M.H. Morkowski S. Laus R. Cancer Res. 2001; 61: 3760-3769PubMed Google Scholar). TRPM8 is gated by cold temperatures, cooling compounds such as menthol and icilin, and positive membrane potentials in a cooperative manner (16McKemy D.D. Neuhausser W.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (1938) Google Scholar, 18Brauchi S. Orio P. Latorre R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15494-15499Crossref PubMed Scopus (281) Google Scholar, 19Voets T. Droogmans G. Wissenbach U. Janssens A. Flockerzi V. Nilius B. Nature. 2004; 430: 748-754Crossref PubMed Scopus (776) Google Scholar, 20Voets T. Owsianik G. Jannssens A. Talavera K. Nilius B. Nat. Chem. Biol. 2007; 3: 174-182Crossref PubMed Scopus (230) Google Scholar). Recent data have identified several lipid messengers like phosphatidylinositol 4,5-bisphosphate (PIP2) or lysophospholipids (LPLs) as mediators of TRPM8 activation (21Rohacs T. Lopes C.M.B. Michailidis I. Logothetis D.E. Nat. Neurosci. 2005; 8: 626-634Crossref PubMed Scopus (484) Google Scholar, 22Vanden Abeele F. Zholos A. Bidaux G. Shuba Y. Thebault S. Beck B. Flourakis M. Panchin Y. Skryma R. Prevarskaya N. J. Biol. Chem. 2006; 281: 40174-40182Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar).Structure-function relations of channel activation are well established for many ion channel families but in the relatively young TRP field, only limited data are available. In a recent study, the S4-S5 region of TRPM8 has been identified as a voltage-sensitive domain that additionally affects the sensitivity to temperature and menthol (20Voets T. Owsianik G. Jannssens A. Talavera K. Nilius B. Nat. Chem. Biol. 2007; 3: 174-182Crossref PubMed Scopus (230) Google Scholar). For TRPM2, the analysis of a C-terminal splice variant detected in neutrophil granulocytes has revealed the importance of single amino acid residues of the NUDT9 domain for ADPR-dependent channel gating (5Wehage E. Eisfeld J. Heiner I. Jüngling E. Zitt C. Lückhoff A. J. Biol. Chem. 2002; 277: 23150-23156Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 23Kühn F.J.P. Lückhoff A. J. Biol. Chem. 2004; 279: 46431-46437Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, no thorough analysis has been undertaken to gain information of how the S5 and S6 regions interact and communicate with the intervening pore region. Hitherto, studies on functional subdomains within the S5-pore-S6 region of TRP channels have concentrated on the cation selectivity determined by charged residues in the pore loop (24Garcia-Martinez C. Morenilla-Palao C. Planells-Cases R. Merino J.M. Ferrer-Montiel A. J. Biol. Chem. 2000; 275: 32552-32558Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Nilius B. Vennekens R. Prenen J. Hoenderop J.G.J. Droogmans G. Bindels R.J.M. J. Biol. Chem. 2001; 276: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 26Nilius B. Prenen J. Janssens A. Owsianik G. Wang C. Zhu M.X. Voets T. J. Biol. Chem. 2005; 280: 22899-22906Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Nilius B. Mahieu F. Prenen J. Janssens A. Owsianik G. Vennekens R. Voets T. EMBO J. 2006; 25: 467-478Crossref PubMed Scopus (247) Google Scholar, 28Voets T. Prenen J. Vriens J. Watanabe H. Janssens A. Wissenbach U. Bodding M. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 33704-33710Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar).The aim of our study was the identification of distinct domains within S5 and S6, common for TRPM2 and TRPM8, which govern interaction with the pore loop and thereby bear functional relevance for both channels. We report that a particular site in the C-terminal end of S6 is essential for the cation versus anion selectivity. By insertion of a lysine into corresponding sites of TRPM2 and TRPM8, channels were created that passed currents in response to the same stimuli as the respective wild-type channels but these currents were no longer carried by cations but by anions.EXPERIMENTAL PROCEDURESMolecular Cloning-For expression in eukaryotic cells, the plasmid constructs pcDNA3-EGFP-TRPM2 and pcDNA3-EGFP-TRPM8 each containing the full-length open reading frame of the corresponding human TRP channel were used. For the generation of the TRPM2/TRPM8 pore chimeras, we introduced recognition sites for AflII (New England Biolabs, Beverly, MA) at corresponding positions of the open reading frame of both channels; one in transmembrane segment S5 and one in S6. Site-directed mutagenesis was performed using the QuikChange mutagenesis system (Stratagene, La Jolla, CA). Defined oligonucleotides were obtained from MWG-Biotech AG (Ebersberg, Germany). The preparation and ligation of the DNA fragments, which have to be exchanged between the two channels was performed as described elsewhere (23Kühn F.J.P. Lückhoff A. J. Biol. Chem. 2004; 279: 46431-46437Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Every point mutation or deletion as well as the correct orientation of the exchanged S5-pore-S6 segments were verified by DNA sequencing with the Big-DYE-Terminator Kit (Perkin Elmer, Roche Applied Sciences, Branchburry, NJ). To exclude the presence of inadvertent mutations in other regions of the channel, two clones with identical results were tested for each chimera or point mutant. All procedures were performed in accordance to the respective manufacturers' instructions, if not indicated otherwise.Cell Culture and Transfection-Chinese ovary hamster cells (CHO-K1) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and cultured in Ham's F12 medium (Biochrome, Berlin, Germany) supplemented with 4 mml-glutamine and 10% (v/v) fetal calf serum (Biochrome). Cells were seeded on glass coverslips at a density of <103 cells/mm2 and grown for 24 h. Subsequently, the pcDNA3-EGFP-TRPM2 or pcDNA3-EGFP-TRPM8 expression constructs were transiently transfected into the CHO cells, using the Trans-Fast transfection reagent (Promega, Mannheim Germany). As controls, cells were transfected with pcDNA3-EGFP vector alone. The transfection procedure was performed as specified by the manufacturer. Patch-clamp measurements were carried out 24 h after transfection in cells visibly positive for EGFP.Electrophysiology-Transfected cells were analyzed with the patch-clamp technique in the conventional whole cell mode, using an EPC 9 equipped with a personal computer with Pulse and X chart software (HEKA, Lamprecht, Germany). The standard bath solution contained (in mm) 140 NaCl, 1.2 MgCl2, 1.2 CaCl2, 5 KCl, 10 HEPES, pH 7.4 (NaOH). For nominally cation-free extracellular conditions, the solution contained 150 mm N-methyl-d-glucamine (NMDG), 10 mm EGTA, and 10 mm HEPES, pH 7.4 (HCl). In some experiments, the standard extracellular bath solution was modified by replacing 140 mm NaCl with either 140 mm sodium glutamate, 140 mm NaBr, 140 mm NaI, or 140 mm NaF, and the reference electrode in the bath solution was a 140 mm NaCl/2% agar salt bridge. The pipette solution contained (in mm) 145 CsCl or 145 Cs-glutamate (as indicated), 8 NaCl, 2 MgCl2, 10 Cs-EGTA, 10 HEPES, pH 7.2 (CsOH). HEPES was replaced with 10 mm MES in experiments in which the pH was 6.0, and 10 mm N-(1,1-dimethyl-2-hydroxy-ethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) at pH 8.8. When a high (1 μm) intracellular Ca2+ concentration was desired, the EGTA concentration in the pipette fluid was reduced to 1 mm, and 0.89 mm CaCl2 was added. For the stimulation of TRPM2 currents, ADP-ribose was added to the pipette solution from a stock, yielding a final concentration of 0.1–1.0 mm. Alternatively, TRPM2 currents were evoked by superfusion of the cells with standard bath solution containing hydrogen peroxide (10 mm; diluted from a 30% stock solution). TRPM8 activation was induced by superfusion with standard bath solution containing 200 μm menthol (Sigma-Aldrich) or 10–30 μm icilin (Cayman Chemical, Ann Arbor, MI). Alternatively, 1.5 μl menthol (from a 200 mm stock solution in Me2SO) or 1.5 μl of Icilin (from a 10–30 mm stock solution in Me2SO) was added directly to the bath chamber (1–2 ml volume). In some experiments, TRPM8 currents were stimulated or suppressed by superfusion with standard bath solution which was cooled immediately before application to ∼4 °C or warmed to 37 °C in a conventional water bath. If not otherwise stated, cells were held at a potential of -60 mV at room temperature. In some experiments, the holding potential was periodically varied (indicated in the figures). The current-voltage relations were obtained during voltage ramps from -90 to +60 mV and back to -90 mV applied over 400 ms. Changes in the liquid junction potential were not quantified in experiments on shifts in the reversal potential evoked by extracellular anion substitution; instead, a comparison was performed between cation selective TRPM channels and S6-Lys mutants during identical bath exchanges.4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, Sigma-Aldrich), used to test inhibition of anion currents, was added from a stock solution (10 mm in water or 50 mm in Me2SO) to the pipette solution or the superfusate (final concentration up to 0.5 mm).N-(p-Amylcinnamoyl) anthranilic acid (ACA; Merck, Germany), used to inhibit currents of wild-type TRPM2, TRPM8, or the M2M8P chimera, was added to the superfusate (final concentration 20 μm, diluted from a 50 mm stock solution in Me2SO).The current density represents the maximal current amplitude (pA) divided by the cell capacitance (pF), a measure of the cell surface. In the case of wild-type TRPM8 and M8-S6-Lys, the values were determined shortly after breaking into the cell and in the case of M2-S6-Lys during plateau current levels in the presence of intracellular ADPR (0.6 mm). Values are given as mean ± S.D. with the number (n) of cells studied.RESULTSTypical characteristics of whole cell currents through the wild-type channels TRPM2 and TRPM8 are shown in Fig. 1 with respect to their activation and their cation selectivity, which proved to be consistent with previous reports (3Peier A.M. Moqrich A. Hergarden A.C. Reeve A.J. Andersson D.A. Story G.M. Earley T.J. Dragoni I. McIntyre P. Bevan S. Patapoutian A. Cell. 2002; 108: 705-715Abstract Full Text Full Text PDF PubMed Scopus (1695) Google Scholar, 4Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (736) Google Scholar, 5Wehage E. Eisfeld J. Heiner I. Jüngling E. Zitt C. Lückhoff A. J. Biol. Chem. 2002; 277: 23150-23156Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 16McKemy D.D. Neuhausser W.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (1938) Google Scholar, 29Sano Y. Inamura K. Miyake A. Mochizuki S. Yokoi H. Matsushime H. Furuichi K. Science. 2001; 293: 1327-1330Crossref PubMed Scopus (382) Google Scholar). CHO cells transiently transfected with TRPM2 developed currents in response to intracellular ADPR (0.1–1.0 mm) that reached the cytosol by diffusion through the patch pipette (Fig. 1A). In standard experiments, Cs+ and glutamate were the main intracellular ions to minimize potential contaminating currents through K+ and Cl- channels (see “Experimental Procedures”). The current-voltage relation of ADPR-induced currents obtained during voltage ramps from -90 to +60 mV showed a reversal potential close to 0 mV, as expected for a nonspecific cation channel (Fig. 1B). Currents in the inward direction were minimized when extracellular Na+ was exchanged by the large impermeable cation NMDG (Fig. 1, A and B), indicating that TRPM2 is a cation channel. Furthermore, TRPM2 currents were sensitive to Ca2+ as previously demonstrated (30McHugh D. Flemming R. Xu S.Z. Perraud A.L. Beech D.J. J. Biol. Chem. 2003; 278: 11002-11006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Removal of extracellular Ca2+ in combination with intracellular Ca2+ buffering with EGTA prevented current development in response to ADPR (data not shown).In patch-clamp experiments performed at room temperature on CHO cells transfected with TRPM8, a basal inward current was apparent that could be suppressed by heating of the cells to 37 °C or could be potentiated by cooling to 4 °C (Fig. 1C). The current-voltage relation shows a pronounced outward rectification and a reversal potential close to 0 mV (Fig. 1D). Again, substitution of extracellular Na+ by NMDG minimized currents in the inward direction without significantly affecting currents in the outward direction (not shown). These data confirm that TRPM8 as well is a non-selective cation channel. TRPM8 currents were not only induced by cold (Fig. 1C) but also by menthol (0.2 mm) or icillin (0.02 mm, not shown). The currents through TRPM8 desensitized rapidly and irreversibly, typically within 1–2 min (Fig. 1C).The S5 and S6 segments of TRPM2 and TRPM8 are quite similar, whereas the central pore sequences display little homology (Fig. 2). For the functional characterization of the extended pore domain, we initially exchanged the almost complete S5-pore-S6 segment of TRPM2 and TRPM8. The sequences at the cut-paste limits of the exchanged S5-pore-S6 segments contained single amino acid substitutions at each end where one hydrophobic amino acid residue was changed to a positively charged lysine (see Fig. 2); this was done to enable the use of the chosen restriction enzyme (see “Experimental Procedures”). The corresponding chimeras M2M8P (TRPM2 containing the S5-pore-S6 segment of TRPM8) and M8M2P (TRPM8 containing the S5-pore-S6 segment of TRPM2) were transiently expressed in CHO cells and characterized with whole cell patch clamp analysis. M8M2P was functionally inactive after stimulation with cold temperatures, menthol, icilin, or ADPR (data not shown). However, cells transfected with the reciprocal chimera M2M8P displayed ADPR-dependent currents (Fig. 3). Infusion of ADPR (0.1–1.0 mm) through the patch pipette into cells kept at a holding potential of -60 mV led to a time-dependent induction of inward currents similar as observed in cells transfected with wild-type TRPM2 (Fig. 3A). However, in contrast to currents of wild-type TRPM2, the inward currents through M2M8P spontaneously and rapidly declined to (mean ± S.D.) 8.2 ± 3.0% of the initial maximum (n = 8; Fig. 3A). To quantify the kinetics, we measured the time over which 90% of the decline had occurred which amounted to 13.4 ± 2.9 s. These inward currents could be restored by depolarization to positive holding potentials. The amplitude of inward currents measured at -60 mV immediately after a depolarizing pulse was positively correlated with the strength of the depolarization (Fig. 3A) and also with its duration (Fig. 3B). The current-voltage relation of ADPR-evoked M2M8P currents (Fig. 3C) was obtained during voltage ramps from -90 to +60 mV, starting from a holding potential of -60 mV. The reversal potential was about -50 mV, considerably more negative if compared with wild-type TRPM2 and TRPM8 (see Fig. 1, B and D). The corresponding reversal potential obtained when the ramp was started from a holding potential of +60 mV was about -30 mV (Fig. 3C). As controls, we tested M2M8P-transfected cells in the absence of ADPR (Fig. 3, C, inset and D) as well as cells transfected with vector construct only (data not shown). Both controls were negative.FIGURE 2Design of TRPM2/TRPM8 channel chimeras. The presumed general structure of TRP channels including the exchanged S5-pore-S6 region (gray shading) is illustrated. The corresponding amino acid sequences of the S5-pore-S6 segments of TRPM2 and TRPM8 are shown in single letter code. Residues conserved between both channels are given in bold. The cut paste limits for chimera construction are marked by asterisks.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Electrophysiological characterization of the channel chimera M2M8P, a TRPM2 channel with the S5-pore-S6 region from TRPM8. A, currents through M2M8P during stepwise changes of the holding potential as indicated in the top panel. The same intracellular solution including ADPR was used as in Fig. 1A. B, time course of currents through M2M8P when a holding potential of -60 mV was applied for 45 s after depolarizing prepulses to +60 mV of variable durations. C, current-voltage relations of M2M8P currents in the presence or in the absence (inset) of ADPR in the patch pipette. The ramps are from the experiments shown in A and D; the times when the individual ramps were recorded are indicated with lowercase letters in A and D. D, whole cell recording of cells expressing M2M8P without stimulation with ADPR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As previously demonstrated (30McHugh D. Flemming R. Xu S.Z. Perraud A.L. Beech D.J. J. Biol. Chem. 2003; 278: 11002-11006Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 31Heiner I. Eisfeld J. Warnstedt M. Radukina N. Jüngling E. Lückhoff A. Biochem. J. 2006; 371: 1045-1053Crossref Google Scholar), intracellular Ca2+ represents a cofactor of ADPR-dependent activation of TRPM2. Therefore, we compared the ADPR-evoked current development of TRPM2 and M2M8P in the absence (10 mm EGTA) and presence of 1 μm Ca2+ in the pipette solution. Similarly to wild-type TRPM2, currents of M2M8P were more strongly stimulated when ADPR was infused in non-saturating concentrations (0.2 mm) along with 1 μm Ca2+ (Fig. 4A) than when ADPR was infused at a low Ca2+ concentration (<10 nm). Moreover, similar M2M8P currents as evoked intracellular ADPR were induced by extracellular H2O2 (10 mm; Fig. 4B). Again, these currents declined at a holding potential of -60 mV and were readily restored by short lasting depolarizations to +60 mV.FIGURE 4Activation and inhibition properties of M2M8P are similar to those of wild-type TRPM2. A, currents in the presence of either high (1 μm) or low (<10 nm) concentrations of intracellular Ca2+ (as indicated) in cells expressing TRPM2 or M2M8P. Currents are normalized to the cell capacitance as well as to the holding potential (thus yielding conductance) because a holding potential of -20 mV instead of -60 mV was used for the large currents through TRPM2 at high Ca2+. B, whole cell currents in cells expressing M2M8P stimulated with hydrogen peroxide (10 mm). Superfusion with H2O2 was started at the time point indicated by the arrow. Note the characteristic delay before the onset of M2M8P current. The holding potential was intermittently set from -60 to +60 mV to restore the declining current amplitudes. C and D, current-voltage relations of M2M8P (C) and wild-type TRPM2 (D) currents obtained in the absence (control) or presence of the inhibitor ACA (20 μm). Cl- was the only intracellular and extracellular anion in these two experiments; see Fig. 5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Recently, it has been demonstrated that N-(p-amylcinnamoyl) anthranilic acid (ACA) is a highly potent and efficient channel blocker of TRPM2, TRPM8, and TRPC6 (32Kraft R. Grimm C. Frenzel H. Harteneck C. Br. J. Pharmacol. 2006; 148: 264-273Crossref PubMed Scopus (117) Google Scholar). Because ACA blocked these channels only from the extracellular side, it has been proposed that it reduces the channel open probability. In our hands, the extracellular application of ACA (20 μm) inhibited ADPR-induced whole cell currents of M2M8P (Fig. 4C) to the same extent as observed for wild-type TRPM2 (Fig. 4D), suggesting that the putative interaction of ACA and the external pore domain is not disturbed in the M2M8P channel chimera. The experiments with ACA were performed with a pipette solution containing Cl- as main anion to avoid block by intracellular glutamate (see next two paragraphs). Thus, with respect to channel activation by ADPR or hydrogen peroxide, co-stimulation by Ca2+ and inhibition by ACA, the gating behavior of M2M8P was not noticeably changed in comparison to wild-type TRPM2.The decline of M2M8P current at negative holding potentials and the restitution of inward currents by depolarizing pulses were consistently found when either glutamate or aspartate (not shown) was present in the pipette solution, at concentrations of 90 mm or more, but not at 40 or 70 mm. To analyze the phenomenon which resembles a voltage-dependent block with a slow time course, we first tested conditions with NMDG as main extracellular cation. These experiments revealed a further and more striking difference of M2M8P in comparison to both, TRPM2 and TRPM8. The characteristic current development of M2M8P (see Figs. 3A and 4B) was not changed by NMDG (Fig. 5A); in fact, NMDG did not elicit any obvious effects on inward or outward currents (compare Figs. 3C, 5B). We examined whether these unexpected results could be explained by permeability to NMDG despite the large size of the NMDG cation, because some channels of the TRP family display partial permeability to NMDG (33Estacion M. Sinkins W.G. Jones S.W. Applegate M.A. Schilling W.P. J. Physiol. 2006; 15: 359-377Crossref Scopus (99) Google Scholar). However, there was no notable shift of the reversal potential of currents t" @default.
• W2090145908 created "2016-06-24" @default.
• W2090145908 creator A5016326642 @default.
• W2090145908 creator A5029976365 @default.
• W2090145908 creator A5053136301 @default.
• W2090145908 date "2007-09-01" @default.
• W2090145908 modified "2023-09-30" @default.
• W2090145908 title "The Transmembrane Segment S6 Determines Cation versus Anion Selectivity of TRPM2 and TRPM8" @default.
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