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• W2081144498 abstract "The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels. The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels. TRPM6 and TRPM7 belong to the TRP channel superfamily (1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 2Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2145) Google Scholar, 3Fleig A. Penner R. Novartis Found. Symp. 2004; 258 (258–266): 248-258Crossref PubMed Google Scholar, 4Schmitz C. Perraud A.L. Fleig A. Scharenberg A.M. Pediatr. Res. 2004; 55: 734-737Crossref PubMed Scopus (30) Google Scholar, 5Montell C. Sci. STKE. 2005; 2005: 1-24Google Scholar) and are distinguished from other known ion channels by virtue of having both ion channel and protein kinase activities (6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar, 7Nadler 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, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 9Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2894-2899Crossref PubMed Scopus (317) Google Scholar, 10Schmitz 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, 11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar). In addition, TRPM6 and TRPM7 uniquely exhibit strong outward rectification, permeation to Ca2+, Mg2+, monovalent cations, and a wide array of trace metals (6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar, 7Nadler 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, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar, 12Monteilh-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). The channel activity of TRPM7 is regulated by intracellular Mg2+ (7Nadler 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) and other divalent cations (13Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 922-927Abstract Full Text Full Text PDF PubMed Google Scholar, 14Kozak J.A. Matsushita M. Nairn A.C. Cahalan M.D. J. Gen. Physiol. 2005; 126: 499-514Crossref PubMed Scopus (114) Google Scholar, 15Kozak J.A. Cahalan M.D. Biophys. J. 2004; 86 (abstr.): 63Google Scholar), Mg2+-ATP (7Nadler 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, 12Monteilh-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, 16Hermosura M.C. Monteilh-Zoller M.K. Scharenberg A.M. Penner R. Fleig A. J. Physiol. 2002; 539: 445-458Crossref PubMed Scopus (165) Google Scholar), phosphatidylinositol 4,5-bisphosphate (14Kozak J.A. Matsushita M. Nairn A.C. Cahalan M.D. J. Gen. Physiol. 2005; 126: 499-514Crossref PubMed Scopus (114) Google Scholar, 17Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (460) Google Scholar), cAMP (18Takezawa 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), and internal and external pH conditions (14Kozak J.A. Matsushita M. Nairn A.C. Cahalan M.D. J. Gen. Physiol. 2005; 126: 499-514Crossref PubMed Scopus (114) Google Scholar, 19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar). Similarly, TRPM6 channel activities have been shown to be inhibited by intracellular Mg2+ and potentiated by external protons (8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar). Recent studies have demonstrated that TRPM6 and TRPM7 are key regulators of Mg2+ homeostasis: mutations of TRPM6 cause familial hypomagnesemia and secondary hypocalcemia (20Walder 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, 21Schlingmann 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); whereas targeted gene deletion of TRPM7 in the DT40 B cell line produced intracellular Mg2+ deficiency and growth arrest (7Nadler 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, 22Schmitz 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). Consistent with its role in Mg2+ and Ca2+ homeostasis, TRPM6 is abundantly expressed in the intestine and the kidney (8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 20Walder 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, 21Schlingmann 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, 23Chubanov V. Gudermann T. Schlingmann K.P. Pflügers Arch. 2005; 451: 228-234Crossref PubMed Scopus (87) Google Scholar), whereas TRPM7 is ubiquitously expressed, with highest expression in the kidney and heart (5Montell C. Sci. STKE. 2005; 2005: 1-24Google Scholar, 6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar). In addition to these channels' regulation of Mg2+ homeostasis, several studies have suggested multiple cellular and physiology functions for TRPM7, including anoxic neuronal death (24Aarts 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 (654) Google Scholar), cell adhesion and actomyosin contractility (25Su 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 (196) Google Scholar, 26Clark 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 (294) Google Scholar), and skeletogenesis (27Elizondo M.R. Arduini B.L. Paulsen J. MacDonald E.L. Sabel J.L. Henion P.D. Cornell R.A. Parichy D.M. Curr. Biol. 2005; 15: 667-671Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Although the mechanisms by which TRPM6 and TRPM7 exert their physiological and/or pathological functions are not yet completely understood, it is clear that permeation of Ca2+ and Mg2+ contributes substantially to the known functions of these channels (7Nadler 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, 20Walder 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, 21Schlingmann 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, 22Schmitz 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, 24Aarts 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 (654) Google Scholar, 25Su 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 (196) Google Scholar, 27Elizondo M.R. Arduini B.L. Paulsen J. MacDonald E.L. Sabel J.L. Henion P.D. Cornell R.A. Parichy D.M. Curr. Biol. 2005; 15: 667-671Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Moreover, a recent study demonstrated that the sensitivity of TRPM7 to external pH may contribute to controlling neurotransmitter release (28Krapivinsky G. Mochida S. Krapivinsky L. Cibulsky S.M. Clapham D.E. Neuron. 2006; 52: 485-496Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Therefore, it is essential to understand the molecular mechanisms underlying the Ca2+ and Mg2+ permeability of TRPM6 and TRPM7, as well as their sensitivities to changes in pH. The aim of the present study was to identify the amino acid residues that determine Mg2+ and Ca2+ permeation of TRPM6 and TRPM7. We previously demonstrated that external protons significantly enhance TRPM6 and TRPM7 inward currents (11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar, 19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar) by decreasing the divalent affinity to the channels. Our results suggested that protons compete with divalents for binding site(s) within the channels' pore. In the present study, we systematically mutated negatively charged amino acid residues within the putative pore-forming region of TRPM7; and identified Glu1047 and Glu1052 of TRPM7 as the key residues that confer divalent selectivity and the sensitivity of the channel to pH. Moreover, we demonstrated that mutations of the equivalent positions (Glu1024 and Glu1029) in TRPM6 produced identical changes, indicating that these two glutamate residues constitute the molecular basis of these channels' Mg2+ and Ca2+ permeability as well as their pH sensitivity. The above findings are critical to understanding the physiological/pathological functions of TRPM6 and TRPM7, and provide molecular insight of the pore architecture of these channels. Molecular Biology—TRPM6 construct was kindly provided by Dr. Joost G. J. Hoenderop. TRPM7 was previously cloned from mouse (6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar). Amino acid substitutions to the pores of TRPM6 and TRPM7 were made using the QuikChange Site-directed Mutagenesis Kit (Stratagene) following the manufacturer's instructions. The primers are shown in supplemental materials Table S1. Functional Expression of TRPM6, TRPM7, and the Mutants—CHOK1 cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 5% CO2. Cells were transiently transfected with wild-type (WT) 5The abbreviations used are:WTwild typeVGCCvoltage-gated Ca2+ channelsDVFdivalent-free solutionNMDGN-methyl-d-glucamineMES4-morpholineethanesulfonic acid. TRPM6, TRPM7, and the mutants of TRPM6 and TRPM7 as previously described (6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar). TRPM7 and its mutants were co-transfected with a green fluorescent protein-containing pTracerCMV2 vector. Electrophysiological recordings were conducted between 36 and 48 h after transfection. Successfully transfected cells were identified by their green fluorescence when illuminated at 480 nm. All patch-clamp experiments were performed at room temperature (20-25 °C). wild type voltage-gated Ca2+ channels divalent-free solution N-methyl-d-glucamine 4-morpholineethanesulfonic acid. Electrophysiology—Whole cell currents were recorded using an Axopatch 200B amplifier. Data were digitized at 10 or 20 kHz, and digitally filtered off-line at 1 kHz. Patch electrodes were pulled from borosilicate glass and fire-polished to a resistance of ∼3 MΩ when filled with internal solutions. Series resistance (Rs) was compensated up to 90% to reduce series resistance errors to <5 mV. Cells in which Rs was >10 MΩ were discarded (29Yue L. Navarro B. Ren D. Ramos A. Clapham D. J. Gen. Physiol. 2002; 160: 845-853Crossref Scopus (128) Google Scholar). For whole cell current recordings, voltage stimuli lasting 250 ms were delivered at 1-5-s intervals, with either voltage ramps or voltage steps ranging from -120 to +100 mV. Unless otherwise stated, following break-in, 3-5 min were allowed to pass to let currents develop to reach a steady-state. A fast perfusion system was used to exchange extracellular solutions, with complete solution exchange achieved in ∼1-3 s (19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar). The internal pipette solution (P1) for whole cell current recordings contained (in mm) 145 cesium methanesulfonate, 8 NaCl, 10 EGTA, and 10 HEPES, with pH adjusted to 7.2 with CsOH. In some experiments (supplementary materials Fig. S1), Mg2+ was added to the pipette solution and the free Mg2+ concentration was titrated to 3 mm (calculated with the MaxChelator software, available at stanford.edu/~cpatton/webmaxcS.htm). In experiments designed to diminish outward currents, pipette solution (P2) contained (mm): NMDG 120, glutamic acid 108, HEPES 10, EGTA 10, CsCl 10, and pH was adjusted to 7.2 with NMDG. The standard extracellular Tyrode's solution contained (mm): 140 NaCl, 5 KCl, 2 CaCl2, 20 HEPES, and 10 glucose, pH adjusted to 7.4 (NaOH). External solutions at various acidic pH were prepared as previously reported (19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar, 30Jordt S.E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (528) Google Scholar, 31Askwith C.C. Wemmie J.A. Price M.P. Rokhlina T. Welsh M.J. J. Biol. Chem. 2004; 279: 18296-18305Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 32Yermolaieva O. Leonard A.S. Schnizler M.K. Abboud F.M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6752-6757Crossref PubMed Scopus (334) Google Scholar). In brief, HEPES (20 mm) was used in the solutions at pH 7.0 and 7.4, and was replaced by 10 mm HEPES and 10 mm MES for the solutions at pH ≤ 6. Divalent-free solution (DVF) contained (mm) 145 NaCl, 20 HEPES, 5 EGTA, 2 EDTA, and 10 glucose, with estimated free [Ca2+] <1nm and free [Mg2+] ≈10 nm at pH 7.4 (calculated with the MaxChelator software). Appropriate Ca2+ or Mg2+ was added to the DVF at pH 7.4 to prepare solutions containing ≤10 μm Mg2+ or Ca2+ (Fig. 5). Solutions containing 0.1, 0.2, 0.5, 1, 2, and 10 mm Mg2+ or Ca2+ were prepared by omitting EDTA and EGTA in the DVF solution, and by adding the appropriate concentrations of Mg2+ or Ca2+, with reductions in Na+ concentration when necessary to maintain constant osmolarity. Isotonic Ca2+ or Mg2+ solution contained 120 mm Ca2+ or Mg2+, 10 mm HEPES, 10 mm glucose, with pH adjusted to pH 7.4. Different cation solutions (Fig. 7) at 30 mm contained (in mm): 30 divalents or monovalents, 20 HEPES, 100 NMDG-Cl, 10 glucose (pH 7.4). Zn2+ solution was prepared at 10 mm due to the low solubility (11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar). All chemical reagents were purchased from Sigma.FIGURE 7Changes in divalent permeability of TRPM7 mutants (mean ± S. E., n = 6). A-E, currents recorded under the indicated conditions were normalized to the current amplitude value obtained in 30 mm Ca2+ external solutions. The sequence of monovalent permeability was not changed (K+ > Cs+ > Na+) in the mutants compared with WT TRPM7; however, the monovalent permeabilities in E1047Q were significantly larger than that of WT TRPM7. F, currents obtained in 30 mm Ca2+ or Mg2+ were normalized to the current amplitude obtained in Tyrode solution. Note that the ratios of ICa/ITyrode and IMg/ITyrode for E1047Q were 0.014 and 0.0096, respectively. The ICa(E1047Q)/ICa(TRPM7) was 0.023, and IMg(E1047Q)/IMg(TRPM7) was 0.011, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ion permeation ratios are typically calculated from reversal potentials (Erev) using Nernst and Goldman-Hodgkin-Katz equations based on the assumption that ions permeate independently and that the electric field in the membrane is constant (33Hille B. Ion Channels of Excitable Membranes.3rd Ed. Sinauer Associates, Inc., Sunderland, MA2003Google Scholar). For TRPM7 channels, however, because the monovalent and divalent cations do not permeate the channels independently (7Nadler 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), it is not adequate to use the Goldman-Hodgkin-Katz equation to estimate the relative permeability (7Nadler 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, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 12Monteilh-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). Thus, the relative permeability was estimated from the inward current amplitude as previously reported (8Voets T. Nilius B. Hoefs S. van der Kemp A.W.C.M. Droogmans G. Bindels R.J.M. Hoenderop J.G.J. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 12Monteilh-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). Data Analysis—Pooled data are presented as mean ± S.E. Dose-response curves were fitted by an equation of the form: E = Emax {1/[1 + (EC50/C)n]}, where E is the effect at concentration C, Emax is the maximal effect, EC50 is the concentration for half-maximal effect, and n is the Hill coefficient (34Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar). EC50 is replaced by IC50 if the effect is an inhibitory effect. Voltage-dependent effects of Ca2+ and Mg2+ on TRPM7 and the mutants were analyzed by fitting the I/I0 ratio curves to the Boltzmann functions: I/I0 = 1/(1 + exp[V0.5 - V]/k) is for the voltage-dependent block, and I/I0 = 1/(1 + exp[V - V0.5]/k) is for the voltage-dependent relief of block; where I0 is the current before and I is the current after application of Mg2+ or Ca2+, V is the membrane potential, V0.5 is the membrane potential at which the current is blocked by 50%, and k is a slope factor representing the voltage dependence of block (35Kerschbaum H.H. Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 2293-2305Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The slope factor k is k = RT/zδF, where z is the valence of blocker and δ is the fraction of the membrane electrical field. Statistical comparisons were made using two-way analysis of variance and two-tailed t test with Bonferroni correction; p < 0.05 indicated statistical significance. E1047Q and E1052Q Substitutions within TRPM7 Pore Alters Its I-V Relationship—When heterologously expressed, TRPM7 constitutes a channel that is characterized by extremely small inward and large outward currents. External divalent cations such as Mg2+ and Ca2+ are permeable to TRPM7 and at the same time block monovalent cations permeating through the pore of the channel. We have previously shown that external protons substantially potentiate TRPM7 inward currents, which may occur through competition of protons with divalent cations for binding sites in the pore of the channel (19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar). To identify potential binding sites for Mg2+, Ca2+, as well as for protons, we systematically exchanged negatively charged residues within the TRPM7 putative pore region with uncharged residues found at equivalent positions in other TRPM family members (Fig. 1). As the increase in inward current induced by pH for TRPM6 is smaller than that observed for TRPM7 (11Li M. Jiang J. Yue L. J. Gen. Physiol. 2006; 127: 525-537Crossref PubMed Scopus (319) Google Scholar, 19Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (154) Google Scholar), we also investigated the contribution of His1039 to the pH sensitivity of TRPM7 because His1039 is replaced by Glu in TRPM6. Therefore, we additionally mutated His1039 to H1039E and H1039M, as TRPM1 and TRPM3 have a Met residue at the equivalent positions, respectively. Within the S5-S6 linker of TRPM7, eight residues were singly or doubly mutated (Fig. 1). The resulting TRPM7 mutants were transiently transfected into CHO-K1 cells and their currents examined for sensitivity to pH and permeability to Mg2+ and Ca2+. Because the endogenous TRPM7-like MIC/MagNuM current is extremely small in CHO-K1 cells (6Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar), the elicited currents obtained upon transfection of the TRPM7 mutants predominantly reflect conductances originating from the expressed TRPM7 pore mutants. Fig. 2 shows currents recorded from various TRPM7 mutants. The current-voltage (I-V) relationships of the mutants D1035N, D1054A, H1039E, and H1039E were similar to that of WT TRPM7. It was surprising that D1054A did not produce a significant change, as this aspartic acid residue is conserved among all TRPM channels. By contrast, the I-V relationships of E1047Q and E1052Q were significantly different from that of WT TRPM7. The inward current of E1052Q was substantially larger than that of WT TRPM7, whereas its outward current was similar to WT TRPM7. E1047Q demonstrated a double rectification I-V profile, with increased inward current and decreased outward current compared with WT TRPM7. The normalized I-V curves of D1035N, D1054A, H1039E, and H1039M were superimposable with that of WT TRPM7, whereas the I-V curves of E1047Q and E1052Q were markedly different from that of WT TRPM7 (Fig. 2H). The average inward and outward current amplitudes obtained for the TRPM7 pore mutants are summarized in Fig. 3A. The ratios of inward currents measured at -120 mV to the outward currents measured at +100 mV of E1047Q and E1052Q were 12- and 8-fold larger than that of WT TRPM7 (Fig. 3B), respectively; indicating that blockade of monovalent inward current by divalent cations was reduced in E1047Q and E1052Q compared with WT TRPM7. The significant changes in current-voltage profiles in E1047Q and E1052Q indicate that E1047 and E1052 are residues critical to TRPM7 channel function.FIGURE 3Average current amplitudes of WT TRPM7 and its mutants. A, mean outward (top) and inward (bottom) current amplitudes measured at +100 and -120 mV, respectively (n = 8). B, ratios of inward versus outward current amplitude (n = 8).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the mutants in which the negatively charged Glu was replaced by positively charged Lys at Glu1047 and Glu1052 positions (E1047K and E1052K), a majority of cells transfected with E1047K and E1052K did not produce measurable currents (data not shown). We were unable to detect expression of the E1052K mutant, suggesting that the amino acid substitution may have affected the overall stability of the protein. However, expression of E1047K was confirmed by Western blot analysis (data not shown), suggesting that either the E1047K mutant is completely inactive or unable to traffic to the cell membrane, thereby indicating that Glu1047 is essential for TRPM7 channel function. Changes in pH Sensitivity in TRPM7 Mutants—For the WT TRPM7 channels, acidification of extracellular bath solution increased the inward current by about 12-fold when the pH was lowered from pH 7.4 to 4.0 (Fig. 4, A1-A2). Similar increases in the inward currents were observed in the H1049E and H1039M mutants (Fig. 4, F1-F2 and G1-G2). The magnitude of the increase in inward current by acidic external bath solutions was considerably smaller for mutants E1052Q, D1035N, and D1054A. However, no significant difference in pH½ (Fig. 4, A3 and C3-G3) was obtained for D1" @default.
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• W2081144498 date "2007-08-01" @default.
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• W2081144498 title "Molecular Determinants of Mg2+ and Ca2+ Permeability and pH Sensitivity in TRPM6 and TRPM7" @default.
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• W2081144498 doi "https://doi.org/10.1074/jbc.m608972200" @default.
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