Activation of TRPM7 Channels by Phospholipase C-coupled Receptor Agonists
2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês
10.1074/jbc.m605300200
ISSN1083-351X
AutoresMichiel Langeslag, Kristopher Clark, Wouter H. Moolenaar, Frank N. van Leeuwen, Kees Jalink,
Tópico(s)Magnesium in Health and Disease
ResumoTRPM7 is a ubiquitously expressed nonspecific cation channel that has been implicated in cellular Mg2+ homeostasis. We have recently shown that moderate overexpression of TRPM7 in neuroblastoma N1E-115 cells elevates cytosolic Ca2+ levels and enhances cell-matrix adhesion. Furthermore, activation of TRPM7 by phospholipase C (PLC)-coupled receptor agonists caused a further increase in intracellular Ca2+ levels and augmented cell adhesion and spreading in a Ca2+-dependent manner (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Regulation of the TRPM7 channel is not well understood, although it has been reported that PIP2 hydrolysis closes the channel. Here we have examined the regulation of TRPM7 by PLC-coupled receptor agonists such as bradykinin, lysophosphatidic acid, and thrombin. Using FRET assays for second messengers, we have shown that the TRPM7-dependent Ca2+ increase closely correlates with activation of PLC. Under non-invasive "perforated patch clamp" conditions, we have found similar activation of TRPM7 by PLC-coupled receptor agonists. Although we could confirm that, under whole-cell conditions, the TRPM7 currents were significantly inhibited following PLC activation, this PLC-dependent inhibition was only observed when [Mg2+]i was reduced below physiological levels. Thus, under physiological ionic conditions, TRPM7 currents were activated rather than inhibited by PLC-activating receptor agonists. TRPM7 is a ubiquitously expressed nonspecific cation channel that has been implicated in cellular Mg2+ homeostasis. We have recently shown that moderate overexpression of TRPM7 in neuroblastoma N1E-115 cells elevates cytosolic Ca2+ levels and enhances cell-matrix adhesion. Furthermore, activation of TRPM7 by phospholipase C (PLC)-coupled receptor agonists caused a further increase in intracellular Ca2+ levels and augmented cell adhesion and spreading in a Ca2+-dependent manner (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Regulation of the TRPM7 channel is not well understood, although it has been reported that PIP2 hydrolysis closes the channel. Here we have examined the regulation of TRPM7 by PLC-coupled receptor agonists such as bradykinin, lysophosphatidic acid, and thrombin. Using FRET assays for second messengers, we have shown that the TRPM7-dependent Ca2+ increase closely correlates with activation of PLC. Under non-invasive "perforated patch clamp" conditions, we have found similar activation of TRPM7 by PLC-coupled receptor agonists. Although we could confirm that, under whole-cell conditions, the TRPM7 currents were significantly inhibited following PLC activation, this PLC-dependent inhibition was only observed when [Mg2+]i was reduced below physiological levels. Thus, under physiological ionic conditions, TRPM7 currents were activated rather than inhibited by PLC-activating receptor agonists. TRPM7 is a ubiquitously expressed nonspecific cation channel that, intriguingly, contains a C-terminal serine-threonine kinase domain. It belongs to the transient receptor potential melastatin-related (TRPM) 2The abbreviations used are: TRPM, TRP (melastatin-related); TRP, transient receptor potential; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BK, bradykinin; EDTA-AM, ethylenediaminetetraacetic acid tetrakis (acetoxymethyl ester); FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; MagNuM, magnesium nucleotide-regulated metal ion current; MIC, magnesium inhibited cation current; PIP2, phosphatidylinositol(4,5)biphosphate; wt, wild type; I/V, current-voltage. subfamily of TRP channels that transduce sensory signals (2Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2173) Google Scholar). TRPM7 functioning appears essential for life in that both knock-out and overexpression of the channels cause growth arrest, loss of cell adhesion, and rapid cell death (3Nadler 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 (813) Google Scholar, 4Schmitz 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 (634) Google Scholar, 5Su L.T. Agapito M.A. Li M. Simonson W.T. Huttenlocher A. Habas R. Yue L. Runnels L.W. J. Biol. Chem. 2006; 281: 11260-11270Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). We recently discovered that low overexpression of TRPM7 induces cell spreading and adhesion and that its activation by PIP2-hydrolyzing receptor agonists leads to the formation of adhesion complexes in a kinase-dependent manner (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Currents carried by TRPM7 channels exogenously expressed in mammalian cells have been analyzed by several groups. In physiological solutions, the channel conducts mainly Ca2+ and Mg2+ (6Voets 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 (537) Google Scholar), but in the absence of these divalent cations, K+ and Na+ (3Nadler 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 (813) Google Scholar, 7Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (638) Google Scholar) permeate efficiently. A characteristic feature is the inhibition of TRPM7 currents by physiological (1–2 mm) intracellular Mg2+ levels; in whole-cell patch clamp experiments, large outwardly rectifying TRPM7 currents (3Nadler 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 (813) Google Scholar, 7Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (638) Google Scholar, 8Kerschbaum H.H. Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 2293-2305Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) are evoked by perfusion with Mg2+-free pipette solutions. Furthermore, MgATP and MgGTP also inhibit the channels (3Nadler 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 (813) Google Scholar, 9Demeuse P. Penner R. Fleig A. J. Gen. Physiol. 2006; 127: 421-434Crossref PubMed Scopus (158) Google Scholar), although some controversy was raised on this issue (10Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar). TRPM7 currents have been termed MagNuM (for Mg2+ nucleotide-regulated metal ion (3Nadler 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 (813) Google Scholar)) or MIC (for Mg2+-inhibited cation (10Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar)) currents. These terms will here be used interchangeably to reflect whole-cell currents evoked by internal Mg2+ depletion. MIC/MagNuM currents revert at about 0 mV and lack voltage- and time-dependent activation (3Nadler 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 (813) Google Scholar, 7Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (638) Google Scholar). Inward currents are predominantly carried by divalent cations, whereas outward currents consist mainly of monovalent cations (at low [Mg2+]i). Outward rectification is most likely due to divalent permeation block of inward currents at negative potentials (3Nadler 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 (813) Google Scholar), because perfusion with divalent-free extracellular solutions augments inward currents and linearizes the I/V relationship. The activation of TRPM7 by internal perfusion with Mg2+-free solutions does not reflect the release of the permeation block, but the precise mechanism has not yet been solved (10Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar). Several reports document that the set point for [Mg2+]i sensitivity is governed by the kinase domain; however, TRPM7 channels lacking the kinase domain can still be activated by internal Mg2+ depletion (4Schmitz 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 (634) Google Scholar, 11Matsushita 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 (160) Google Scholar, 12Schmitz 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 (164) Google Scholar). Thus, the interactions of TRPM7 with Mg2+ are complex; Mg2+ is conducted through the channel pore, causes voltage-dependent permeation block, and influences gating at the cytosolic surface. The exact mechanisms by which receptor agonists regulate TRPM7 are less well characterized, and the published data are, at least partly, conflicting. An initially claimed indispensable role for the kinase domain (7Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (638) Google Scholar) was challenged in subsequent studies (4Schmitz 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 (634) Google Scholar, 11Matsushita 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 (160) Google Scholar, 12Schmitz 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 (164) Google Scholar, 13Takezawa 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 (166) Google Scholar). We have recently shown (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar) that the TRPM7 α-kinase specifically phosphorylates the heavy chain of myosin-II, thereby strongly influencing cell adhesion. Importantly, association with and subsequent phosphorylation of myosin-II depend on prior activation of the channel by phospholipase C (PLC)-coupled receptors, and influx of extracellular Ca2+ constitutes an essential step in this process (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). In accordance with this, TRPM7 binds directly to several PLC isoforms, including PLCγ and PLCβ (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (463) Google Scholar). The stimulatory effect of PLC on TRPM7 observed in intact cells by biochemical, cell-biological, and live cell-imaging studies contrasts with a report that, in human embryonic kidney 293 (HEK293) cells, whole-cell TRPM7 currents are inhibited by PIP2 hydrolysis (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (463) Google Scholar). PIP2-dependent gating also occurs in other TRP family members, including TRPV1 (15Prescott E.D. Julius D. Science. 2003; 300: 1284-1288Crossref PubMed Scopus (482) Google Scholar), TRPM4 (16Zhang Z. Okawa H. Wang Y. Liman E.R. J. Biol. Chem. 2005; 280: 39185-39192Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Nilius B. Mahieu F. Prenen J. Janssens A. Owsianik G. Vennekens R. Voets T. EMBO J. 2006; 25: 467-478Crossref PubMed Scopus (252) Google Scholar) TRPM5 (18Liu D. Liman E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15160-15165Crossref PubMed Scopus (357) Google Scholar), and TRPM8 (19Liu B. Qin F. J. Neurosci. 2005; 25: 1674-1681Crossref PubMed Scopus (271) Google Scholar, 20Rohacs T. Lopes C.M. Michailidis I. Logothetis D.E. Nat. Neurosci. 2005; 8: 626-634Crossref PubMed Scopus (498) Google Scholar). Finally, Takezawa and colleagues (13Takezawa 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 (166) Google Scholar) recently reported that, in HEK-293 cells (expressing only endogenous muscarinic receptors), carbachol attenuated TRPM7 currents via the Gs-cAMP signaling pathway, whereas PLC activation was not involved. Given the discrepancies between the documented whole-cell patch clamp results and our cell biological observations, we re-examined how PLC-activating receptor agonists affect TRPM7 currents using non-invasive techniques in cells that moderately overexpress TRPM7. To this end, we combined fluorescence resonance energy transfer (FRET) assays for second messengers with perforated patch experiments and Ca2+ fluorometry to show that opening of TRPM7 channels closely correlates with PLC activation but not with cAMP/cGMP signaling. We also showed that, in perforated patches, TRPM7 currents are evoked by treatment of intact cells with a membrane-permeable Mg2+ chelator (EDTA-AM) and that these currents are inhibited rather than augmented by PLC-activating receptor agonists. We conclude that PLC-coupled agonists activate rather than inhibit TRPM7 in intact cells. Materials—Amphotericin B, MgATP, bradykinin, lysophosphatidic acid, spermine, La(NO3)3, Gd2(CO3)3, sodium nitroprusside, prostaglandin E1, and niflumic acid were from Sigma. Oregon Green 488 BAPTA-1 AM, Fura Red AM, Indo-1 AM, o-nitrophenyl-EGTA AM, pluronic F127, EDTA-AM, and BAPTA-AM were from Molecular Probes. Ionomycin, 2-aminoethoxydiphenyl-borate (2-APB), SKF 96365, isobutylmethylxanthine, forskolin, and thapsigargin were from Calbiochem-Novabiochem. Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, streptomycin, and neomycin were from Invitrogen, and FuGENE 6 was from Roche Diagnostics. Constructs—TRPM7 constructs were as described previously (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). The PIP2 FRET sensor (eCFP-PHδ1 and eYFP-PHδ1) was previously generated in our laboratory (21van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Sensors for cAMP and cGMP were kind gifts from Dr. M. Zaccolo (University of Padova, Padova, Italy) and Dr. W. Dostmann (University of Vermont, Burlington, VT), respectively, and Yellow Cameleon 2.1 was a gift from Dr. R. Tsien (University of California-San Diego, La Jolla, CA). Immunostaining and Kinase Reaction—A TRPM7 antibody was raised to amino acids 1748–1862 in rabbits as detailed in Ref. 1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar. Preimmune serum was used as a control in all experiments. For immunolabeling, cells were fixed with 4% paraformaldehyde in phosphate buffer, permeabilized with 0.1% Triton X-100, and incubated with rabbit anti-TRPM7 sera (1:200) followed by horseradish peroxidase-conjugated anti-rabbit Ig (1:1000). Amplification was by tyramide-conjugated fluorescein isothiocyanate (PerkinElmer Life Sciences). For the in vitro kinase assay, TRPM7 was precipitated from lysed cells with anti-TRPM7 antibodies and assayed for kinase activity as published recently (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Cell Culture, Fluorimetric Experiments—Culture of mouse N1E-115 and Phoenix packaging cells was as described previously (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Ratiometric and pseudoratiometric Ca2+ recordings on cells on glass coverslips were carried out essentially as published in Refs. 21van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 22Rasmussen U. Broogger C.S. Sandberg F. Acta Pharm. Suec. 1978; 15: 133-140PubMed Google Scholar, 23Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1990; 265: 12232-12239Abstract Full Text PDF PubMed Google Scholar in HEPES-buffered saline, pH 7.2, at 37 °C. All traces were calibrated with ionomycin and BAPTA as published previously (23Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1990; 265: 12232-12239Abstract Full Text PDF PubMed Google Scholar). Dynamic FRET essays were carried out as described previously (21van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 24Ponsioen B. Zhao J. Riedl J. Zwartkruis F. van der Krogt G. Zaccolo M. Moolenaar W.H. Bos J.L. Jalink K. EMBO Rep. 2004; 5: 1176-1180Crossref PubMed Scopus (361) Google Scholar). Excitation was at 425 nm using an ND3 filter, and cyan and yellow fluorescent protein emissions were collected simultaneously at 470 ± 20 and 530 ± 25 nm, respectively. Data were acquired at 4 Hz, and FRET was expressed as the ratio of cyan to yellow fluorescent protein signals. This ratio was set to 1.0 at the onset of the experiments, and changes are expressed as the percent of deviation from this initial value. Patch Clamp Experiments—Electrophysiological recordings were collected using the HEKA EPC9 system. Current recordings were digitized at 100 kHz (ramp and block pulse protocols) or 10 Hz (steady-state whole-cell currents). Borosilicate glass pipettes were fire-polished to 2–4 MΩ. After establishment of the GΩ seal, the patched membrane was ruptured by gentle suction to obtain whole-cell configuration, or amphotericin B (240 μg/ml) was used to obtain the perforated patch configuration with the typical access resistance of 3–10 MΩ. Solutions were (in mm) 120 whole-cell pipette potassium-glutamate, 30 KCl, 1 MgCl2, 0.2 CaCl2, 1 EGTA, 10 HEPES, pH 7.2, and 1 MgATP; 140 external solution NaCl, 5 KCl, 0–1 MgCl2, 0–10 CaCl2, 10 HEPES, and 10 glucose adjusted to pH 7.3 with NaOH; for perforated patch recordings, the pipette solution was complemented with 240 μg/ml amphotericin B, and MgATP was omitted. TRPM7 Expression Raises Basal and Agonist-induced Cytosolic Ca2+ Levels—Consistent with previous observations (3Nadler 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 (813) Google Scholar), we found that transient overexpression of TRPM7 channels was lethal to N1E-115 cells and several other cell lines tested within a few days (data not shown). Furthermore, at early time points after transfection, much of the TRPM7 protein was found localized to biosynthetic compartments. To circumvent these problems, we expressed TRPM7 in N1E-115 cells at low levels by retroviral transduction (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). These cells, termed N1E-115/TRPM7 cells, were viable, divided normally, and could be routinely kept in culture for several months (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). Using a TRPM7-specific antibody (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar), the expression level in N1E-115/TRPM7 cells was found to be 2–3 times higher than that in the parental cells (Fig. 1a). Under these conditions, the TRPM7 mainly localized to the plasma membrane, as shown in Fig. 1b. Moreover, depletion of intracellular Mg2+ in whole-cell patch clamp recordings showed that N1E-115/TRPM7 has ∼2.5 times higher current density of MIC/MagNuM currents than wild type (wt) cells (supplemental Fig. S1). Ratiometric analysis (see "Experimental Procedures") showed that intracellular Ca2+ was elevated significantly in N1E-115/TRPM7 cells (108.9 ± 6.0 nm, n = 50) as compared with parental cells (85.3 ± 2.5 nm, n = 50; p < 0.05) (Fig. 1c). Thus, TRPM7 channel expression contributes to basal Ca2+ homeostasis, in agreement with reports that the channels are partly preactivated (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar, 4Schmitz 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 (634) Google Scholar, 11Matsushita 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 (160) Google Scholar, 25Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 922-927Abstract Full Text Full Text PDF PubMed Google Scholar, 26Hanano T. Hara Y. Shi J. Morita H. Umebayashi C. Mori E. Sumimoto H. Ito Y. Mori Y. Inoue R. J. Pharmacol. Sci. 2004; PubMed Google Scholar). Because cell biological and biochemical data (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar) indicate that TRPM7 channels are activated by bradykinin (BK), we monitored the effect of the addition of BK on TRPM7-mediated Ca2+ influx by comparing N1E-115/TRPM7 to parental cells. In parental cells, 1 μm BK induced a cytosolic Ca2+ increase (765.9 ± 9.7 nm, n = 80) that peaked within seconds and subsequently returned to values close to resting levels (92.9 ± 2.9 nm) within 60 s (Fig. 1d, left panel). This corresponds to a mean increase of 7.6 nm from basal levels, which was not statistically significant. In contrast, in N1E-115/TRPM7 cells, the BK-induced transient Ca2+ increase (peak 843.3 ± 12.7 nm) was followed by a prominent sustained Ca2+ elevation (141.0 ± 7.0 nm, n = 120) that lasted for several minutes before returning to resting levels rather abruptly (Fig. 1d, right panel; quantification in Fig. 1c). This corresponds to a mean increase in sustained Ca2+ levels of 32.1 nm (p < 0.001, paired t test) in accordance with the higher TRPM7 expression levels in these cells. Thus, in N1E-115/TRPM7 cells, the addition of BK elicits a sustained phase in the Ca2+ response, in good agreement with our cell biological data (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar). In summary, the observed increase in Ca2+ influx is mediated by TRPM7 channels and is not due to the retroviral transduction procedure (supplemental Fig. S2). TRPM7 Activation Is Downstream of PLC in Intact N1E-115 Cells—Endogenous B2 bradykinin receptors in N1E-115 cells signal predominantly via the Gq-linked PLC pathway (27Coggan J.S. Thompson S.H. Am. J. Physiol. 1995; 269: C841-C848Crossref PubMed Google Scholar), but some reports suggest that, depending on the cell type, the B2 receptors may occasionally either inhibit (28Hanke S. Nurnberg B. Groll D.H. Liebmann C. Mol. Cell. Biol. 2001; 21: 8452-8460Crossref PubMed Scopus (31) Google Scholar) or stimulate (29Albert O. Ancellin N. Preisser L. Morel A. Corman B. Life Sci. 1999; 64: 859-867Crossref PubMed Scopus (7) Google Scholar) the production of cAMP. We set out to identify the signaling events responsible for BK-induced activation of TRPM7 in N1E-115/TRPM7 cells by correlating Ca2+ fluorometry with the activation of intracellular signaling pathways, as detected by various FRET assays. BK caused rapid breakdown of a significant fraction (60–80%) of the plasma membrane PIP2 pool, as detected by a FRET assay (21van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) that reports membrane PIP2 content (Fig. 2a, upper panel, first trace). The Gq/PLC-coupled receptor agonist lysophosphatidic acid and thrombin receptor-activating peptide also activate PLC, although to a lesser extent (∼20–30% of BK values; n > 200) (Fig. 2a, upper panel, second and third trace (21van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar)). Similar to BK, the initial Ca2+ peak induced by these agonists was followed by sustained Ca2+ influx in N1E-115/TRPM7 cells (Fig. 2a, lower panel). In contrast, sustained Ca2+ influx was not seen when cells were stimulated with agonists of receptors that do not couple to PLC, such as prostaglandin E1 (Fig. 2a, 4th trace, and data not shown). Thus, TRPM7 activation correlates well with PLC activation/Ca2+ signaling. To address the possible involvement of cAMP in the BK-induced opening of TRPM7, we monitored cAMP levels in intact N1E-115/TRPM7 cells using a genetically encoded cAMP sensor (30Zaccolo M. De Giorgi F. Cho C.Y. Feng L. Knapp T. Negulescu P.A. Taylor S.S. Tsien R.Y. Pozzan T. Nat. Cell Biol. 2000; 2: 25-29Crossref PubMed Scopus (396) Google Scholar, 31Bacskai B.J. Hochner B. Mahaut-Smith M. Adams S.R. Kaang B.K. Kandel E.R. Tsien R.Y. Science. 1993; 260: 222-226Crossref PubMed Scopus (436) Google Scholar). The addition of BK to N1E-115/TRPM7 cells had no effect on cAMP levels (n = 8), whereas forskolin (25 μm) readily raised cAMP levels (Fig. 2b, left panel). Furthermore, pretreatment of cells with pertussis toxin to specifically inhibit Gi and thereby block receptor-induced decreases in cAMP levels did not affect the BK-induced Ca2+ influx (data not shown). Therefore, in N1E-115/TRPM7 cells, changes in cAMP levels do not mediate the BK-induced opening of TRPM7. In addition, neither prostaglandin E1, which activates Gs to cause a rapid and sustained increase in [cAMP]i (Fig. 2b, middle panel), nor sphingosine-1-phosphate (S1P, data not shown), which couples to Gi and G13 but not to PLC in N1E-115 cells (32Postma F.R. Jalink K. Hengeveld T. Offermanns S. Moolenaar W.H. Curr. Biol. 2001; 11: 121-124Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), had any effect on Ca2+ levels. We conclude that there is no evidence for a role of cAMP in BK-mediated Ca2+ influx in N1E-115/TRPM7 cells. We also investigated the effects of the nitric oxide donor nitroprusside, because nitric oxide was reported to activate TRPM7-mediated Ca2+ influx in cultured cortical neurons (33Aarts M. Iihara K. Wei W.L. Xiong Z.G. Arundine M. Cerwinski W. MacDonald J.F. Tymianski M. Cell. 2003; 115: 863-877Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). In intact parental and N1E-115/TRPM7 cells, nitroprusside triggered the production of cGMP (Fig. 2c, left panel (24Ponsioen B. Zhao J. Riedl J. Zwartkruis F. van der Krogt G. Zaccolo M. Moolenaar W.H. Bos J.L. Jalink K. EMBO Rep. 2004; 5: 1176-1180Crossref PubMed Scopus (361) Google Scholar)) without affecting [Ca2+]i (Fig. 2c, right panel). In conclusion, TRPM7 opening closely correlates with PLC activation but not with other G protein-linked signals. Activation of PLC Inhibits Whole-cell TRPM7 Currents in N1E-115/TRPM7 Cells—In whole-cell patch clamp experiments using HEK-293 cells overexpressing M1 muscarinic receptors, TRPM7 channels were shown to be inhibited by carbachol-induced PIP2 breakdown (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (463) Google Scholar). This inhibition was reverted to by intracellular perfusion with a water-soluble PIP2 analogue. However, our cell biological observations (1Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (297) Google Scholar) and Ca2+ data (see above) show that PLC activation causes opening rather than closure of TRPM7 channels. We therefore tested whether voltage ramp-induced TRPM7 currents, recorded in whole cells with Mg2+-free pipet
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