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Role of the α-Kinase Domain in Transient Receptor Potential Melastatin 6 Channel and Regulation by Intracellular ATP

2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês

10.1074/jbc.m800167200

ISSN

1083-351X

Autores

Stéphanie Thebault, Gang Cao, Hanka Venselaar, Qi Xi, René J.M. Bindels, Joost G.J. Hoenderop,

Tópico(s)

Saffron Plant Research Studies

Resumo

Transient receptor potential melastatin 6 (TRPM6) plays an essential role in epithelial Mg2+ transport. TRPM6 and its closest homologue, TRPM7, both combine a cation channel with an α-kinase domain. However, the role of this α-kinase domain in TRPM6 channel activity remains elusive. The aim of this study was to investigate the regulation of TRPM6 channel activity by intracellular ATP and the involvement of its α-kinase domain. We demonstrated that intracellular Na- and Mg-ATP decreased the TRPM6 current in HEK293 cells heterogeneously expressing the channel, whereas Na-CTP or Na-GTP had no effect on channel activity. Whole cell recordings in TRPM6-expressing HEK293 cells showed that deletion of the α-kinase domain prevented the inhibitory effect of intracellular ATP without abrogating channel activity. Mutation of the conserved putative ATP-binding motif GXG(A)XXG (G1955D) in the α-kinase domain of TRPM6 inhibited the ATP action, whereas this effect remained preserved in the TRPM6 phosphotransferase-deficient mutant K1804R. Mutation of the TRPM6 autophosphorylation site, Thr1851, into either an alanine or an aspartate, resulted in functional channels that could still be inhibited by ATP. In conclusion, intracellular ATP regulates TRPM6 channel activity via its α-kinase domain independently of α-kinase activity. Transient receptor potential melastatin 6 (TRPM6) plays an essential role in epithelial Mg2+ transport. TRPM6 and its closest homologue, TRPM7, both combine a cation channel with an α-kinase domain. However, the role of this α-kinase domain in TRPM6 channel activity remains elusive. The aim of this study was to investigate the regulation of TRPM6 channel activity by intracellular ATP and the involvement of its α-kinase domain. We demonstrated that intracellular Na- and Mg-ATP decreased the TRPM6 current in HEK293 cells heterogeneously expressing the channel, whereas Na-CTP or Na-GTP had no effect on channel activity. Whole cell recordings in TRPM6-expressing HEK293 cells showed that deletion of the α-kinase domain prevented the inhibitory effect of intracellular ATP without abrogating channel activity. Mutation of the conserved putative ATP-binding motif GXG(A)XXG (G1955D) in the α-kinase domain of TRPM6 inhibited the ATP action, whereas this effect remained preserved in the TRPM6 phosphotransferase-deficient mutant K1804R. Mutation of the TRPM6 autophosphorylation site, Thr1851, into either an alanine or an aspartate, resulted in functional channels that could still be inhibited by ATP. In conclusion, intracellular ATP regulates TRPM6 channel activity via its α-kinase domain independently of α-kinase activity. Within the transient receptor potential family of cation channels, three members from the Melastatin subgroup, TRPM6, 3The abbreviations used are: TRPM, transient receptor potential melastatin; GFP, green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; AMP·PNP, adenosine 5′-(β,γ-imino)triphosphate. TRPM7, and TRPM2, display unique primary structures known as "chanzymes," i.e. fusion of an ion channel poreforming region with an enzymatic domain (1Nadler M.J. Hermosura M.C. Inabe K. Perraud A.L. Zhu Q. Stokes A.J. Kurosaki T. Kinet J.P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (802) Google Scholar, 2Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar, 3Ryazanov A.G. FEBS Lett. 2002; 514: 26-29Crossref PubMed Scopus (127) Google Scholar, 4Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 5Scharenberg A.M. Pfluegers Arch. Eur. J. Physiol. 2005; 451: 220-227Crossref PubMed Scopus (47) Google Scholar). Remarkably, the proteins TRPM6 and TRPM7 share 52% sequence identity and play a crucial role in Mg2+ homeostasis (6Montell C. Curr. Biol. 2003; 13: R799-R801Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Konrad M. Schlingmann K.P. Gudermann T. Am. J. Physiol. 2004; 286: F599-F605Crossref PubMed Scopus (194) Google Scholar). Mutations in the TRPM6 gene are responsible for the autosomal recessive form of familiar hypomagnesemia with secondary hypocalcemia (8Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. Syrrou M. Nielsen S. Sassen M. Waldegger S. Seyberth H.W. Konrad M. Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (647) Google Scholar, 9Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (470) Google Scholar), whereas a missense mutation in TRPM7 is responsible for the pathogenesis of Guamanian neurodegenerative disorders (10Hermosura M.C. Nayakanti H. Dorovkov M.V. Calderon F.R. Ryazanov A.G. Haymer D.S. Garruto R.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11510-11515Crossref PubMed Scopus (172) Google Scholar). In addition, the lethal phenotype caused by inactivation of the TRPM7 gene in lymphocytes cells can be rescued by extracellular Mg2+ supplementation (1Nadler M.J. Hermosura M.C. Inabe K. Perraud A.L. Zhu Q. Stokes A.J. Kurosaki T. Kinet J.P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (802) Google Scholar, 11Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Although the physiological activation mechanism of TRPM6 remains unknown, there is evidence of a constitutive activity in heterologous expression systems. TRPM6 displays a nonselective cation current conducted by divalent ions inwardly and monovalent ions outwardly and exhibits a steep outwardly rectifying current-voltage relation (12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). A recent study reported that epidermal growth factor hormone acts as an autocrine/paracrine magnesiotropic hormone, specifically stimulating TRPM6 current via engagement of its receptor at the basolateral membrane of distal convoluted tubule cells (13Groenestege W.M. Thebault S. van der Wijst J. van den Berg D. Janssen R. Tejpar S. van den Heuvel L.P. van Cutsem E. Hoenderop J.G. Knoers N.V. Bindels R.J. J. Clin. Investig. 2007; 117: 2260-2267Crossref PubMed Scopus (295) Google Scholar). Previous studies showed that both TRPM6 and TRPM7 currents are inhibited by millimolar concentrations of intracellular Mg2+ (1Nadler M.J. Hermosura M.C. Inabe K. Perraud A.L. Zhu Q. Stokes A.J. Kurosaki T. Kinet J.P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (802) Google Scholar, 11Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). In rat basophilic leukemia and Jurkat T cells, a native conductance with properties similar to cells heterogeneously expressing TRPM7 was described and termed magnesium nucleotide-dependent metal cation channel (14Hermosura M.C. Monteilh-Zoller M.K. Scharenberg A.M. Penner R. Fleig A. J. Physiol. 2002; 539: 445-458Crossref PubMed Scopus (165) Google Scholar) or Mg2+-inhibited cation channel (15Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar, 16Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (267) Google Scholar). Subsequent studies reported critical amino acids in the pore regions of TRPM7 (Glu1047/Glu1052) and TRPM6 (Glu1024/Glu1029) that play a role in Mg2+ and pH sensitivity (17Li M. Du J. Jiang J. Ratzan W.J. Su L.T. Runnels L.W. Yue L. J. Biol. Chem. 2007; 282: 25817-25830Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The functional α-kinase domain at the carboxyl terminus of TRPM7 has been structurally characterized by x-ray crystallography (4Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In contrast to the classical kinases, the α-kinase domain displays unique features including a zinc finger domain as well as a region involved in the catalysis of ATP-triggered reactions (18Ryazanov A.G. Pavur K.S. Dorovkov M.V. Curr. Biol. 1999; 9: R43-R45Abstract Full Text Full Text PDF PubMed Google Scholar). Despite this information, the role of intracellular ATP on the regulation of channel activity remains controversial (19Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 922-927Abstract Full Text Full Text PDF PubMed Google Scholar, 20Gwanyanya A. Sipido K. Vereecke J. Mubagwa K. Am. J. Physiol. 2006; 291: C627-C635Crossref PubMed Scopus (62) Google Scholar, 21Monteilh-Zoller M.K. Hermosura M.C. Nadler M.J. Scharenberg A.M. Penner R. Fleig A. J. Gen. Physiol. 2003; 121: 49-60Crossref PubMed Scopus (431) Google Scholar, 22Demeuse P. Penner R. Fleig A. J. Gen. Physiol. 2006; 127: 421-434Crossref PubMed Scopus (156) Google Scholar). The current consensus is that ATP acts as an intracellular Mg2+ chelator, which decreases the concentration of intracellular free Mg2+, resulting in increased TRPM7-mediated conductance (19Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 922-927Abstract Full Text Full Text PDF PubMed Google Scholar, 20Gwanyanya A. Sipido K. Vereecke J. Mubagwa K. Am. J. Physiol. 2006; 291: C627-C635Crossref PubMed Scopus (62) Google Scholar, 21Monteilh-Zoller M.K. Hermosura M.C. Nadler M.J. Scharenberg A.M. Penner R. Fleig A. J. Gen. Physiol. 2003; 121: 49-60Crossref PubMed Scopus (431) Google Scholar, 22Demeuse P. Penner R. Fleig A. J. Gen. Physiol. 2006; 127: 421-434Crossref PubMed Scopus (156) Google Scholar). These observations were challenged by a study demonstrating that both Mg2+ and Mg2+-nucleotides act in synergy to inhibit TRPM7 activity via two distinct binding sites located within the carboxyl terminus (22Demeuse P. Penner R. Fleig A. J. Gen. Physiol. 2006; 127: 421-434Crossref PubMed Scopus (156) Google Scholar). Another debated aspect concerns the role of the α-kinase in modulation of channel activity. Some studies reported that phosphotransferase-deficient TRPM7 mutants showed a reduced sensitivity for intracellular Mg2+ (11Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar), exhibited a reduced current amplitude upon G protein-coupled receptor stimulation (23Takezawa R. Schmitz C. Demeuse P. Scharenberg A.M. Penner R. Fleig A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6009-6014Crossref PubMed Scopus (164) Google Scholar), or were even not functional (2Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar), whereas others found that TRPM7 lacking the α-kinase domain displayed similar Mg2+ sensitivity compared with the wild-type channel (24Matsushita M. Kozak J.A. Shimizu Y. McLachlin D.T. Yamaguchi H. Wei F.Y. Tomizawa K. Matsui H. Chait B.T. Cahalan M.D. Nairn A.C. J. Biol. Chem. 2005; 280: 20793-20803Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In contrast to the TRPM7 α-kinase domain little is known about the TRPM6 α-kinase domain. Recently, TRPM6 activity has been shown to be controlled by RACK1 via its interaction with the α-kinase (25Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). An additional study reported that the α-kinase of TRPM6 is capable of cross-phosphorylation of TRPM7 but not vice versa, underlying the functional nonredundancy of these two chanzymes (26Schmitz C. Dorovkov M.V. Zhao X. Davenport B.J. Ryazanov A.G. Perraud A.L. J. Biol. Chem. 2005; 280: 37763-37771Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In this line, Li et al. (27Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar) demonstrated that TRPM6, TRPM7, and TRPM6/7 are three distinct ion channels that exhibit different functional characteristics, thereby showing unequivocally that TRPM6 can form by itself functional channels without TRPM7 co-expression. Although the previously reported data suggested that the ATP effect on TRPM7 is independent of α-kinase activity, there are no potential domains identified outside of the α-kinase domain mediating a direct interaction with ATP. Notably, renal distal convoluted tubule cells contain a large number of mitochondria per unit length of any cell along the nephron, underling a dynamic capacity of intracellular ATP (28Madsen K.M. V. J. Tisher C.C. J. Electron. Microsc. Tech. 1988; 9: 187-208Crossref PubMed Scopus (41) Google Scholar). Given the predominant expression pattern of TRPM6 in the distal convoluted tubule (12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar), the molecular mechanism of ATP-regulated TRPM6 channel activity was investigated. Cell Culture and Transfection—HEK293 cells were grown in Dulbecco's modified Eagle's medium (Bio Whittaker-Europe, Verviers, Belgium) containing 10% (v/v) fetal calf serum, 2 mm l-glutamine, and 10 μg/ml ciproxin at 37 °C in a humidity-controlled incubator with 5% (v/v) CO2. The cells were transiently transfected with the respective constructs using Lipofectamine 2000 (Invitrogen), as described previously (29Trouet D. Nilius B. Voets T. Droogmans G. Eggermont J. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 632-638Crossref PubMed Scopus (62) Google Scholar), and electrophysiological recordings were performed 48 h after transfection. The transfected cells were identified by their green fluorescence when illuminated at 480 nm. Nontransfected (GFP-negative) cells from the same batch were used as controls. DNA Constructs—The bicistronic expression vector pCINeo-IRES-GFP containing the full-length cDNA of aminoterminally hemagglutinin (HA)-tagged human TRPM6 (GenBank™ accession number NM_017662, 1 μg of plasmid DNA/million cells (12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) was used to co-express TRPM6 and enhanced GFP in HEK293 cells. Δkinase (L1749X), ATP-TRPM6 (G1955D), TRPM6 phosphotransferase-deficient (K1804R and D1933A), TRPM6-T1851A (T1851A) and TRPM6-T1851D (T1851D) were generated using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Following mutagenesis, the mutated pCINeo-IRES-GFP-hTRPM6 constructs were digested with EcoRI, resulting in a ∼1-kb fragment containing the mutation that was subcloned with its corresponding region in the original pCINeo-IRES-GFP-hTRPM6 construct. The wild-type α-kinase domain of TRPM6 as well as the G1955D mutants were subcloned in GST vectors using PCR. All of the constructs were verified by sequencing analysis. The primer sequences used are: Δkinase (forward, 5′-gaggagagttccccttaaaaccttgataaaagc-3′; backward, 5′-gcttttatcaaggttttaaggggaactctcctc-3′), K1804R (forward, 5′-ggacaagttttcattgtcaggtcctttcttcctgaggtt-3′; backward, 5′-aacctcaggaagaaaggacctgacaatgaaaacttgtcc-3′), D1933A (forward, 5′-gagaaaatttgacagctccatctgttataaaac-3′; backward, 5′-gttttataacagatggagctgtcaaattttctc-3′), T1851(A/D) (forward, 5′-ccaagtgaaaccacaagccataccctacacacc-3′; backward, 5′-ggtgtgtagggtatggcttgtggtttcacttgg-3′), and ATP-TRPM6 (forward, 5′-ggaccggccaatttggacgaagatgcaattaga-3′; backward, 5′-tctaattgcatcttcgtccaaattggccggtcc-3′). Electrophysiology and Solutions—Patch clamp experiments were performed in the tight seal whole cell configuration at room temperature (20–25 °C) using an EPC-10 patch clamp amplifier computer controlled by PatchMaster software (HEKA Electronik, Lambrecht, Germany). Patch pipettes had resistances between 2 and 5 mΩ after filling with the standard intracellular solution. The cells were held at 0 mV, and voltage ramps of 200-ms duration ranging from –100 to +100 mV were applied every 2 s. Extracting the current amplitudes at –80 and +80 mV from individual ramp current records assessed the temporal development of membrane currents. Current densities were obtained by normalizing the current amplitude to the cell membrane capacitance. The extracellular solutions contained 150 mm NaCl, 10 mm HEPES, and 1 mm CaCl2 (titrated to pH 7.4 with NaOH). The standard pipette solution contained 150 mm NaCl, 10 mm Na-EDTA, and 10 mm HEPES (titrated to pH 7.2 with NaOH). In experiments testing nucleotide effect, intracellular solutions were supplemented with Na-ATP, Mg-ATP, Na-GTP, or Na-CTP as indicated in the legends. In some experiments, Na-EDTA was replaced by Na-HEDTA (10 mm). CaBuf software (by G. Droogmans, KU Leuven, Leuven, Belgium) was used to estimate the free Mg2+ concentration. The osmolarity of the solutions was adjusted to ∼310 mOsm/kg with mannitol. All of the chemicals were purchased from Sigma. Immunoblotting—72 h after transfection, HEK293 cells were lysed, and protein samples were denatured by incubation for 30 min at 37 °C in Laemmli buffer and then subjected to SDS-PAGE (50 μg/lane). Immunoblots were incubated with the mouse anti-HA (Sigma) antibody. Subsequently, the blots were incubated with sheep horseradish peroxidase-conjugated anti-mouse IgG (Sigma) and then visualized using an enhanced chemiluminescence system. In Vitro Phosphorylation Assays—72 h after transfection, HEK293 cells were lysed for 1 h on ice in lysis buffer (150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.5 adjusted by NaOH, 1% (v/v) Nonidet P-40 including the protease inhibitors leupeptin (0.01 mg/ml), pepstatin (0.05 mg/ml), and phenylmethylsulfonyl fluoride (1 mm)). After centrifugation of the lysates, supernatants were incubated with anti-HA antibody immobilized on protein A-agarose beads (Kem-En-Tec A/S, Copenhagen, Denmark) for 3 h at room temperature. Following three washing steps with lysis buffer, the beads were incubated in a total volume of 30 μl of kinase reaction buffer (50 mm HEPES, pH 7.4 adjusted by KOH, 4 mm MnCl2, 0.5 mm CaCl2, 100 μm ATP) and 2 μCi of [γ-32P]ATP for 30 min at 30 °C. After 30 min of TRPM6 phosphorylation the reaction was terminated by three washing steps with phosphorylation washing buffer (50 mm HEPES, pH 7.4 adjusted by KOH, 4 mm MnCl2, 0.5 mm CaCl2). Phosphorylation was analyzed after gel electrophoresis by autoradiography. Expression and Purification of GST Fusion Proteins—GST-fused proteins were expressed according to the manufacturer's protocol (Amersham Biosciences). To purify the GST fusion proteins, the cell pellets were lysed in cold lysis buffer (50 mm Tris, pH 7.4 adjusted by NaOH, 120 mm NaCl, 0.5% (v/v) Nonidet P-40, 1 mm EDTA, 0.5 mg/ml lysozyme including the protease inhibitors leupeptin (0.01 mg/ml), pepstatin (0.05 mg/ml), and phenylmethylsulfonyl fluoride (1 mm)) for 40 min and subsequently centrifuged for 30 min at 4 °C. The supernatant was added directly to glutathione-Sepharose 4B beads (Amersham Biosciences) for purification. After 2 h of incubation at room temperature, the beads were washed extensively with lysis buffer. The bound proteins were eluted with SDS-PAGE loading buffer, separated on SDS-polyacrylamide gel, and detected by Coomassie staining. ATP Binding Assay—GST and GST-kinase fusion proteins were purified as described above, and subsequently ∼100 ng was incubated with 2 μCi of [γ-32P]ATP in 50 μl of ATP binding buffer (45 mm HEPES (13Groenestege W.M. Thebault S. van der Wijst J. van den Berg D. Janssen R. Tejpar S. van den Heuvel L.P. van Cutsem E. Hoenderop J.G. Knoers N.V. Bindels R.J. J. Clin. Investig. 2007; 117: 2260-2267Crossref PubMed Scopus (295) Google Scholar), 10 mm EDTA, 0.9 mm EGTA, 0.14 mm KCl, 9% v/v glycerol, 0.018% v/v Nonidet P-40, and 0.12 mg of bovine serum albumin/ml) for 20 min at 37 °C. After three wash steps with ATP binding buffer in the absence of [γ-32P]ATP, the samples were analyzed by scintillation counting. Sequence Analysis and Structure Modeling—A structural model of the TRPM6 α-kinase domain was built based on the crystal structure of the TRPM7 α-kinase domain (4Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) (Protein Data Bank code 1IA9 (AMP·PNP complex)) using the WHAT IF/Yasara Twinset (31Krieger E. Koraimann G. Vriend G. Proteins. 2002; 47: 393-402Crossref PubMed Scopus (1175) Google Scholar). The standard parameter settings were used. The sequence alignment and all other relevant modeling information is available online (supplemental Figs. S1 and S2). Data Analysis—In all experiments, the data are expressed as the means ± S.E. Overall statistical significance was determined by analysis of variance. In case of significance, differences between the means of two groups were analyzed by unpaired t test. p < 0.05 was considered significant. Statistical analysis was performed using the SPSS software (SPSS Inc., Chicago, IL). Effect of Intracellular ATP on TRPM6 Channel Activity—To investigate the functional properties of TRPM6, electrophysiological measurements were performed on transiently transfected HEK293 cells expressing HA-TRPM6. These cells developed large whole cell currents that reached their maximal amplitude (260 ± 30 and –10 ± 1.5 pA/pF at +80 and –80 mV, respectively) within a 200-s time period. These currents were not observed in mock transfected cells (Fig. 1A). TRPM6 displayed a characteristic outwardly rectifying current-voltage relation (Fig. 1B). Moreover, as already described (12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar), outwardly rectifying currents were also observed in mock transfected HEK293 cells dialyzed with a Mg2+-free pipette solution (containing 10 mm EDTA). However, when compared with TRPM6-transfected cells, these currents developed over a longer time period and reached maximal amplitudes that are ∼10 times smaller (Fig. 1A). Next, in the presence of 10 mm Na-ATP and no added Mg2+ in the patch pipette solution, TRPM6 currents activated slowly and reached its maximal amplitude after 200 s (Fig. 1, C and D, 102 ± 25 and –5.9 ± 1.5 pA/pF at +80 and –80 mV, respectively). This maximal amplitude was 2.5-fold reduced in comparison with the wild-type current. Perfusion of higher Na-ATP concentrations (12 mm) abrogated its development (Fig. 1, C and D). Because ATP is well known to sequester free intracellular Mg2+ (32Wang M. Tashiro M. Berlin J.R. J. Physiol. 2004; 555: 383-396Crossref PubMed Scopus (41) Google Scholar), it was essential to determine whether the inhibitory Na-ATP effect results from Mg2+ buffering or from direct inhibition of channel activity. Considering that TRPM6 is inhibited by increased intracellular Mg2+ levels (12Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar), the inhibitory effect of ATP cannot be due to its Mg2+ chelator property. However, to clarify this point, we performed the experiments in the presence of 10 mm EDTA, a strong Mg2+ chelator (free [Mg2+]i, ∼0 mm as calculated with CaBuf program). Importantly, the addition of Na-ATP (2 mm) in the presence of 10 mm EDTA significantly reduced the TRPM6-mediated current at both +80 and –80 mV (Fig. 1, E and F, –46 ± 6% and –53 ± 2%, respectively, p < 0.05, n = 17–22 cells). Furthermore, replacing EDTA with HEDTA in the patch pipette solution did not affect TRPM6 activity (supplemental Fig. S3). As summarized in Fig. 1G, after breaking the membrane (0 s), TRPM6 current amplitude was increased in the presence of EDTA compared with Na-ATP. In the presence of 10 mm EDTA, the addition of either 2 mm Na-ATP 10 mm Na-ATP or 12 mm Na-ATP in the patch pipette reduced TRPM6 current development in comparison with recordings with only 10 mm EDTA. These data indicate that free ATP inhibits TRPM6 activity independently of the intracellular Mg2+ concentration. To further confirm this hypothesis, we substituted Na-ATP with Mg-ATP, a physiological form of ATP in the patch pipette solution. In these experiments the intracellular Mg2+ concentration was clamped at micromolar concentrations with 10 mm EDTA. Our data showed that Na-ATP and Mg-ATP inhibit TRPM6 currents in a similar dose-dependent manner with a comparable IC50 of ∼1.3 mm (Fig. 1H). Specificity and Reversibility of the ATP Effect on TRPM6—To evaluate the specificity of intracellular ATP on TRPM6 activity, TRPM6-transfected HEK293 cells were perfused with Na+-nucleotide trisphosphates. Neither Na-GTP nor Na-CTP was able to affect the TRPM6 current compared with the nucleotide-free controls, whereas Na-ATP inhibited the current at +80 and –80 mV (Fig. 2). Na+-nucleotides trisphosphates did not affect the current in mock transfected cells. Relevance of the α-Kinase Domain for TRPM6 Channel Activity—To investigate the role of α-kinase domain in the inhibitory ATP effect, the α-kinase domain was truncated by introducing a stop codon at position 1749 (Δkinase; see Fig. 4A). This Δkinase mutant was subsequently analyzed for its auto-phosphorylation activity. In vitro phosphorylation experiments revealed that truncation of the α-kinase domain abolished kinase activity in contrast to the wild-type channel (Fig. 3A, top panel). Immunoblot analysis indicated equal expression of both TRPM6 and Δkinase (Fig. 3A, lower panel). Next, the Δkinase mutant was functionally characterized and its sensitivity to Na-ATP was studied. HEK293 cells expressing Δkinase displayed currents with lower amplitude compared with the wild-type channel at both +80 and –80 mV (55 ± 14% of the wild type remained, p < 0.05, n = 16–19 cells) but with similar activation kinetics (Fig. 3, B and C). The current-voltage relation of Δkinase is comparable with wild-type TRPM6 (Figs. 1B and 3F). Na-ATP (2 mm) did not affect either the current amplitude at 200 s (Fig. 3, D and E) or the current-voltage relation of the Δkinase mutant (Fig. 3F), indicating the importance of the α-kinase in the inhibitory ATP effect. Subsequently, the role of this domain was studied by site-directed mutagenesis (Fig. 4A). First, based on sequence homology, the mutations rendering TRPM7 phosphotransferase-deficient (K1648A and D1775A (11Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar)) were introduced in TRPM6 (positions 1804 and 1933 for K1804R and D1933A, respectively). Second, the Thr1851 that has been recently identified as an autophosphorylated amino acid residue within the α-kinase (25Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) was either point-mutated into an alanine, resulting in a putative nonphosphorylated mutant (T1851A) or into an aspartate, mimicking a constitutive auto-phosphorylated mutant (T1851D). Using in vitro phosphorylation experiments, the phosphotransferase-deficient mutants, D1933A and K1804R showed an abolished α-kinase activity in contrast to the wild-type channel (Fig. 4B, top panel). Furthermore, the T1851A and T1851D mutants exhibited reduced auto-phosphorylation in comparison with the wild-type channel (Fig. 4B, top panel). Immunoblot analysis indicated comparable expression of the (mutated) TRPM6 proteins (Fig. 4B, bottom panels). Next, all of the mutants were functionally characterized in whole cell recordings. HEK293 cells expressing the phosphotransferase-deficient mutant K1804R exhibited current amplitudes similar to the wild-type channel, whereas the phosphotransferase-deficient D1933A mutant had no activity (Fig. 4, C and D). In addition, both T1851A (nonphosphorylated) and T1851D (constitutively phosphorylated) mutants resulted in active channels with comparable current amplitudes as wild-type TRPM6 (Fig. 4, C and D). Subsequently, Na-ATP was tested on the functional TRPM6 mutants. Na-ATP perfusion reduced both inward and outward K1804R-, T1851A-, and T1851D-mediated currents (37 ± 4, 36 ± 7, and 55 ± 6% of the current remained at +80 mV, respectively; Fig. 5, A–F). No effect of Na-ATP was observed on the current-voltage relations (Fig. 5, G–I).FIGURE 3Effect of ATP on TRPM6 Δ kinase. A, Δkinase mutant was immunoprecipitated, subjected to in vitro protein kinase assay and analyzed by autoradiography in comparison with the wild type (top panel). The expression of TRPM6 and Δkinase mutant was verified by immunoblotting (bottom panel). Representative results of three independent experiments are shown. B and C, averaged time course of current density from HEK293 cells transfected with Δkinase (inverted triangles) when dialyzed with standard pipette solution in comparison with TRPM6 (open circles) at +80 (B) and –80 mV (C). D and E, application of 2 mm ATP in the pat

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