TRPM6 Forms the Mg2+ Influx Channel Involved in Intestinal and Renal Mg2+ Absorption
2003; Elsevier BV; Volume: 279; Issue: 1 Linguagem: Inglês
10.1074/jbc.m311201200
ISSN1083-351X
AutoresThomas Voets, Bernd Nilius, Susan Hoefs, Annemiete W.C.M. van der Kemp, Guy Droogmans, René J.M. Bindels, Joost G.J. Hoenderop,
Tópico(s)Magnesium in Health and Disease
ResumoMg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia. Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia. Mg2+ is the fourth most abundant cation in the body and the second most common cation in the intracellular fluid. Homeostasis of Mg2+ levels is tightly regulated and depends on the balance between intestinal absorption and renal excretion. The kidney provides the most sensitive control for the Mg2+ balance. About 80% of the total serum Mg2+ is ultrafiltered through the glomerular membrane and subsequently reabsorbed in consecutive segments of the nephron (1.Dai L.J. Ritchie G. Kerstan D. Kang H.S. Cole D.E. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (267) Google Scholar). The final urinary excretion of Mg2+ is mainly determined by the active reabsorption of Mg2+ in the distal convoluted tubule (DCT), 1The abbreviations used are: DCT, distal convoluted tubule; DM-nitrophen, dimethoxynitrophenamine tetrasodium salt; HEK, human embryonic kidney; HSH, hypomagnesemia with secondary hypocalcemia; MagNuM, Mg2+-nucleotide-inhibited; MIC, Mg2+-inhibited cation; NCC, NaCl co-transporter; NMDG+, N-methyl-d-glucosamine; RR, ruthenium red; TRP, transient receptor potential. because virtually no reabsorption takes places beyond this segment (1.Dai L.J. Ritchie G. Kerstan D. Kang H.S. Cole D.E. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (267) Google Scholar). Recently, a positional candidate screening approach in consanguineous families with hypomagnesemia with secondary hypocalcemia (HSH) revealed a critical region identified on chromosome 9q21.13 (2.Schlingmann 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, 3.Walder 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). Individuals suffering from HSH display neurologic symptoms including seizures and tetany during infancy. These symptoms can be suppressed by life-long dietary magnesium supplementation, but, untreated, the disease may be fatal or result in neurological damage. The patho-physiology of HSH is largely unknown, but physiological studies have shown that there are defects in both intestinal Mg2+ absorption and renal Mg2+ reabsorption. Subsequent analysis of the critical region pointed to a gene, TRPM6, which was mutated in patients with HSH (2.Schlingmann 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, 3.Walder 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). The TRPM6 protein is a member of the transient receptor potential channel (TRP) family (4.Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Based on the structural and sequence similarities between individual TRP proteins, three subgroups are distinguished, namely the canonical TRPC-, the vanilloid-like TRPV-, and the melastatin-like TRPM subfamilies. Most members of the TRPC- and TRPV-subfamilies have been characterized as Ca2+-permeable cation channels playing a role in Ca2+ homeostasis and signaling (4.Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). However, the functional characterization of TRPM proteins is much more incomplete. TRPM6 shows 50% sequence homology with TRPM7 (also known as TRP-PLIK), which forms a Ca2+ and Mg2+-permeable cation channel. Unlike other members of the TRP family, TRPM6 and TRPM7 contain long carboxyl-terminal domains with similarity to the α-kinases (5.Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar). The identification of TRPM6 as the gene mutated in HSH represents the first case in which a human disorder has been attributed to a channel kinase. The aim of the present study was to functionally characterize TRPM6 as the first molecularly identified protein involved in active Mg2+ (re)absorption. To this end, the (sub)localization of TRPM6 was investigated by immunohistochemical analysis of kidney and duodenum sections. Subsequently, human TRPM6 cDNA was cloned, transfected into human embryonic kidney (HEK-293) cells and characterized using patch-clamp analysis in combination with Magfura-2-based intracellular Mg2+ measurements. Taken together, this study indicates that TRPM6 is a Mg2+-permeable channel localized to the apical domain of the distal convoluted tubule and the brush-border membrane of the absorptive cells in duodenum, the main sites of transepithelial Mg2+ transport. The absence of the TRPM6-dependent Mg2+ influx pathway in patients with HSH provides a straightforward explanation for the severe hypomagnesemia. Immunohistochemistry—Antiserum against TRPM6 was obtained by immunization of guinea pigs with a peptide encoding the carboxyl-terminal 15 amino acids of mouse TRPM6 (NH2-ERDKNRSSLEDHTRL-COOH) coupled to key hole limpet hemocyanin. Kidneys from C57Bl/6 mice were cut into pieces, placed in 1% (w/v) periodate-lysine-paraformaldehyde fixative for 2 h, incubated overnight in phosphate-buffered saline containing 15% (w/v) sucrose, and frozen in liquid nitrogen. Duodenum samples from C57Bl/6 mice were cut into pieces, rinsed with phosphate-buffered saline, and frozen immediately in liquid nitrogen. Subsequently, kidney and duodenum samples were cut into 5–7-μm sections and used for different staining procedures as described previously (6.Hoenderop J.G. Hartog A. Stuiver M. Doucet A. Willems P.H. Bindels R.J. J. Am. Soc. Nephrol. 2000; 11: 1171-1178Crossref PubMed Google Scholar). The antibody against parvalbumin was purchased from Swant (Bellinzona, Switzerland), whereas the NaCl co-transporter (NCC) antibody was kindly donated by Dr. J. Loffing (Zurich, Switzerland) (7.Nijenhuis T. Hoenderop J.G. Loffing J. van der Kemp A.W. Van Os C. Bindels R.J. Kidney Int. 2003; 64: 555-564Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). All negative controls, including sections incubated with pre-immune serum and undergoing pre-absorption for 1 h with 10 μg/ml TRPM6-peptide or solely with conjugated secondary antibodies, were devoid of any staining. Photographs were taken with a Bio-Rad MRC 1000 confocal laser-scanning microscope. Electrophysiology, Intracellular Mg2+Measurements, and Mg2+Uncaging—HEK-293 cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, 2 mm l-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 10% CO2. They were transiently transfected with human TRPM6 in the pCINeo/IRES-GFP vector using previously described methods (8.Trouet 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 made between 16 and 36 h after transfection. Successfully transfected cells were identified by their green fluorescence when illuminated at 480 nm. Patch-clamp experiments were performed in the tight seal whole-cell configuration at room temperature (20–25 °C) using an EPC-9 patch-clamp amplifier and Pulse software (HEKA Electronics). Currents were digitized at 10 kHz and digitally filtered at 2.9 kHz. Unless noted otherwise, cells were held at 0 mV, and voltage ramps of 200 ms duration ranging from –150 to 100 mV were applied every 2 s. Current densities were obtained by normalizing the current amplitude to the cell membrane capacitance. The time course of current development was determined by measuring the current in at 80 and –80 mV. The standard pipette solution contained 150 mm NaCl, 10 mm EDTA, and 10 HEPES mm, pH 7.2. Where indicated, NaCl was equimolarly substituted by NMDG-Cl. In some experiments, the EDTA was omitted, and 0.1 mm EGTA was added. The extracellular solutions contained 150 mm NaCl and 10 mm HEPES, pH 7.4, supplemented with either 10 mm EDTA (divalent-free solutions) or the indicated concentrations of divalent cations. To measure the relative permeability of divalent cations, we used solutions containing 120 mm NMDG-Cl and 10 mm HEPES, pH 7.4, supplemented with 20 mm of the chloride salt of the respective divalent cations. The pipette solution used for the intracellular Mg2+ measurements (e.g. Fig. 3a) contained 150 mm NaCl, 0.1 mm EGTA, 0.2 mm Magfura-2, and 10 mm HEPES, pH 7.2. To measure the intracellular Mg2+ concentration, the dye was excited with light alternated between 350 and 380 nm using a monochromator-based system (TILL Photonics, Planegg, Germany), and the fluorescence signal was measured using a photo-diode. The ratio R of the fluorescent signal at both wavelengths was converted into intracellular Mg2+ concentration using the Grynkiewicz equation (9.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). In vivo calibration constants were obtained by measuring R in cells dialyzed with pipette solutions containing 0, 2, and 100 mm free Mg2+. The pipette solution used in the Mg2+ uncaging experiments contained 150 mm NaCl, 5 mm DM-nitrophen, 4.5 mm MgCl2, 1 mm Magfura-2, and 10 mm HEPES, pH 7.2. The estimated free Mg2+ concentration of this solution is 0.05 mm. Stepwise increases in intracellular Mg2+ were obtained by applying short flashes of UV light from a xenon arc flash lamp (Rapp OptoElectronics, Hamburg, Germany). We also performed experiments using a similar DM-nitrophen-containing pipette solution devoid of Mg2+. Under these conditions, UV flashes had no significant effect on the whole-cell currents in TRPM6-expressing cells, thereby excluding direct effects of UV light or photolysis products of DM-nitrophen. Data Analysis—Data analysis and display was done using Microcal Origin software, version 7.0 (OriginLab Corporation). Group data are presented as mean ± S.E. Student's t test and one-way analysis of variance (ANOVA) were used for statistical comparisons between means. The recent finding that the TRPM6 messenger is found in the kidney and small intestine (3.Walder 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, 2.Schlingmann 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), prompted us to determine the (sub)cellular localization of the TRPM6 protein in these organs. Using antiserum raised against the last 15 amino acids of the mouse TRPM6 carboxyl-terminus (NH2-ERDKNRSSLEDHTRL-COOH), immunopositive staining was observed in the superficial cortex of the mouse kidney, whereas the outer and inner medulla were negative. Importantly, TRPM6 was predominantly localized to the apical membrane of the immunopositive tubules (Fig. 1a). Next, co-stainings were performed combining the antisera against TRPM6 and the thiazide-sensitive NCC, which is a specific marker for DCT, the main site of active transcellular Mg2+ reabsorption along the nephron (10.Obermuller N. Bernstein P. Velazquez H. Reilly R. Moser D. Ellison D.H. Bachmann S. Am. J. Physiol. 1995; 269: F900-F910PubMed Google Scholar). A complete co-localization of TRPM6 and NCC was observed in mouse kidney, as illustrated in Fig. 1a. The segmental distribution of TRPM6 was compared with that of parvalbumin and calbindin-D28K, two cytosolic proteins that can act as endogenous Ca2+ and Mg2+ buffers (11.Yang W. Lee H.W. Hellinga H. Yang J.J. Proteins. 2002; 47: 344-356Crossref PubMed Scopus (110) Google Scholar). TRPM6 co-localized with parvalbumin in the proximal part of DCT (DCT1; Fig. 1b) and with calbindin-D28K in the distal part of DCT (DCT2; Fig. 1c). Expression of calbindin-D28K extended to TRPM6-negative cells in the connecting tubule and the cortical collecting duct (Fig. 1c). These results demonstrate that TRPM6 expression is restricted to DCT, where it is localized along the apical membrane (Fig. 1d). Interestingly, Schlingmann et al. reported the presence of TRPM6 mRNA in the proximal tubule (2.Schlingmann 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). In the present study we did not, however, detect TRPM6 protein expression in this segment. Because TRPM6 mRNA was detected in the previous study by PCR using microdissected tubules, it is possible that small impurities of the dissected segments have been sufficient to yield a weak PCR band. In small intestine, TRPM6 was abundantly present in brush-border membranes of duodenum (Fig. 1e). Absorptive epithelial cells in the villi stained intensively for TRPM6. No staining was observed when duodenum sections were incubated with pre-immune anti-serum (Fig. 1e, right). For electrophysiological analysis of TRPM6, HEK-293 cells were transiently transfected with a vector containing the coding sequence for human TRPM6 (GenBank™ accession number NM_017662). Transfected cells perfused with a NaCl-based extracellular solution containing 1 mm CaCl2 exhibited characteristic outwardly rectifying currents immediately upon establishment of the whole-cell configuration (Fig. 2, a and c). When dialyzed with a divalent-free internal solution containing 10 mm EDTA, the amplitude of these outwardly rectifying currents rapidly increased to reach a plateau level within 100–200 s (Fig. 2a). These outwardly rectifying currents showed little or no time dependence during voltage steps to different potentials (Fig. 2d). In agreement with previous reports (12.Monteilh-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, 13.Nadler 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), we also observed outwardly rectifying currents in non-transfected HEK-293 cells dialyzed with a Mg2+-free internal solution. However, when compared with TRPM6-expressing cells, these currents were undetectable immediately upon establishment of the whole-cell configuration, developed over a longer time period, and reached maximal amplitudes that were ∼10 times smaller (Fig. 2b). HEK-293 cells transfected with two of the TRPM6 mutants identified in HSH patients (TRPM6S590X and TRPM6Arg-736 fs X-737, in which fs stands for frameshift and X is a stopcodon) displayed currents with similar amplitude and activation kinetics as non-transfected cells (Fig. 2, a and b), indicating that these mutant proteins are non-functional. Note that all mutations found in HSH patients code for truncated proteins that lack all six transmembrane domains as well as the carboxyl-terminal α-kinase domain (2.Schlingmann 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, 3.Walder 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). We next investigated the ionic nature of the TRPM6-induced currents. Using NaCl-based extracellular solutions containing low millimolar concentrations of CaCl2 and/or MgCl2, the outwardly rectifying TRPM6-induced currents reversed close to 0 mV (e.g. Fig. 2, c and e). Substitution of extracellular Cl– by aspartate did not alter the reversal potential or amplitude of the outward currents (data not shown), excluding a significant contribution of Cl– fluxes to the observed currents. Likewise, substitution of extracellular Na+ by the large cation N-methyl-d-glucosamine (NMDG+) did not change the shape of the current-voltage relation or the inward current amplitude; with both Mg2+ and Ca2+ at 2 mm in the extracellular solution, the inward current density at –80 mV was –13.6 ± 2.2 pA/pF and –13.7 ± 2.3 pA/pF (n = 4) in the presence of 150 mm Na+ and NMDG+, respectively. These results indicate that the inward current is almost exclusively carried by Mg2+ and/or Ca2+. However, large inward currents were observed upon removal of all extracellular divalent cations, yielding slightly inwardly rectifying currents that reversed close to 0 mV with identical NaCl-based solutions on either side of the membrane (Fig. 2e). Subsequent substitution of extracellular Na+ by NMDG+ virtually abolished inward currents, shifting the reversal potential to voltages more negative than –50 mV (Fig. 2e). Thus, in the absence of divalent cations, the TRPM6-induced channel becomes highly permeable to Na+. Outward currents were strongly reduced when NMDG+ was the sole cation in the intracellular solution, indicating that the large outward currents observed with the NaCl-based intracellular solutions are mainly carried by Na+ (Fig. 2f). Thus, the outwardly rectifying current-voltage profile of the TRPM6-induced current measured with physiological concentrations of extracellular divalent cations reflects the small inward flux of divalent cations and the large outward flux of monovalent cations. Given that TRPM6 is expressed in Mg2+-absorbing epithelia and that mutations in the TRPM6 gene cause HSH, we further tested the permeability of the TRPM6-induced channel to Mg2+ and other divalent cations. Significant inward currents were measured with all tested divalent cations, each acting as the sole charge carrier in the extracellular solution (Fig. 3a). At a concentration of 20 mm, the permeation rank order determined from the inward current amplitude at –80 mV was Ba2+ ≥ Ni2+ > Mg2+ > Ca2+ (Fig. 3a, inset). When normalized to the inward current amplitude in 20 mm Ca2+, we obtained relative values of 2.02 ± 0.18, 1.81 ± 0.22, and 1.33 ± 0.09 for Ba2+, Ni2+, and Mg2+, respectively (n = 4–7). The divalent cations had a differential blocking effect on the outward currents, and the rank order of blocking efficiency was Ni2+ > Mg2+ > Ca2+ > Ba2+ (Fig. 3a). We also estimated permeability ratios (PX/PNa) based on the reversal potentials (Erev) measured with the respective divalent cations (each 20 mm) in the extracellular solution and 150 mm intracellular Na+. This procedure yielded a different permeation sequence (respective PX/PNa values in parentheses), namely Ni2+ (9.2 ± 0.3) > Mg2+ (9.0 ± 0.4) > Ca2+ (6.9 ± 0.3) > Ba2+ (6.2 ± 0.2). It is, for two reasons, questionable whether these PX/PNa values adequately describe the permeability properties of TRPM6. First, the slope of the current-voltage relation is very shallow around Erev in the presence of extracellular divalent cations. Exact measurements of Erev are therefore difficult and error prone, because small changes in background current can have significant effects on Erev. Second, the calculation of PX/PNa from Erev is based on the Goldman-Hodgkin-Katz equations, which assume that ions permeate independently and that the electric field in the membrane is constant (14.Hille B. Ion Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA2001Google Scholar, 12.Monteilh-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). However, the outward rectification of the TRPM6-induced current is much stronger than predicted by these equations, which makes the validity of these assumptions highly questionable. To further substantiate the Mg2+ permeability of the TRPM6-induced channel, we measured changes in the intracellular Mg2+ concentration directly using the Mg2+-sensitive ratiometric dye Magfura-2. In these experiments, cells were dialyzed with a Magfura-2-containing pipette solution devoid of Mg2+ and EDTA (see “Experimental Procedures”), which resulted in basal intracellular Mg2+ concentrations between 0.05 and 0.1 mm (Fig. 3b). The addition of 5 mm Mg2+ to the extracellular solution, which contained 2 mm Ca2+ during the entire course of the experiment, resulted in a significant increase of the intracellular Mg2+ concentration in TRPM6-expressing cells (Fig. 3b). Intracellular Mg2+ was increased further with higher extracellular Mg2+ concentrations or when the plasma membrane was hyperpolarized to –80 mV (Fig. 3b), consistent with Mg2+ influx through the TRPM6-induced channel. It is generally assumed that the luminal concentration of free Mg2+ in the Mg2+-absorbing part of the nephron is in the range of 0.2– 0.7 mm (1.Dai L.J. Ritchie G. Kerstan D. Kang H.S. Cole D.E. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (267) Google Scholar). To preferentially conduct Mg2+ in the presence of Ca2+, which is present at a concentration of ∼1mm, the apical Mg2+ influx pathway should exhibit a higher affinity for Mg2+ than for Ca2+. As a first approach to investigating whether the TRPM6-induced current satisfies this requirement, we compared the sensitivity of monovalent TRPM6-induced currents to Mg2+ and Ca2+. As shown in Fig. 3c, inward Na+ currents were blocked in a voltage-dependent manner by low micromolar concentrations of both Mg2+ and Ca2+. However, Mg2+ blocked the inward currents at lower concentrations than Ca2+ (Fig. 3, c and d). The dose-response curves for block of Na+ current at –80 mV yielded KD values of 1.1 and 4.8 μm and Hill coefficients of 0.85 and 0.83 for Mg2+ and Ca2+, respectively. The most straightforward explanation of the difference in KD values between Mg2+ and Ca2+ is that Mg2+ binds with a higher affinity to a binding site in the channel pore. Alternatively, the inhibitory effect of divalent cations on the permeation of monovalent cations might be due to a regulatory divalent cation binding site outside the pore, but this is highly unlikely given the strong voltage dependence of the block by Mg2+ and Ca2+. Thus, although the relative contribution of Mg2+ and Ca2+ to the inward current under physiological conditions is hard to estimate and expected to vary with changes in their respective extracellular concentrations, these data indicate that the pore of the TRPM6-induced channel has a higher affinity for Mg2+ than for Ca2+. This contrasts with all known Ca2+-selective channels (15.Hess P. Tsien R.W. Nature. 1984; 309: 453-456Crossref PubMed Scopus (578) Google Scholar, 14.Hille B. Ion Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA2001Google Scholar), including members of the TRP superfamily (16.Hoenderop J.G. Vennekens R. Muller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. J. Physiol. 2001; 537: 747-761Crossref PubMed Scopus (229) Google Scholar), which generally display a 10–1000 times lower affinity for Mg2+ than for Ca2+. Monovalent currents were highly sensitive to ruthenium red (RR), a potent blocker of several members of the TRPV family (17.Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7090) Google Scholar, 18.Nilius B. Prenen J. Vennekens R. Hoenderop J.G. Bindels R.J. Droogmans G. Br. J. Pharmacol. 2001; 134: 453-462Crossref PubMed Scopus (104) Google Scholar). As illustrated in Fig. 3e, 10 μm RR caused a strong inhibition of inward monovalent currents while leaving outward currents unaltered. Fig. 3f summarizes the voltage and concentration dependence of the block of monovalent currents by RR. The RR concentration for half-maximal current inhibition increased from ∼100 nm at –120 mV to >10 μm at potentials more positive than –20 mV. Such voltage dependence is generally interpreted as a direct block of the channel pore within the transmembrane electrical field. Interestingly, we found that inward currents carried by 10 mm Mg2+ were significantly less sensitive to inhibition by RR (40 ± 7% block at –80 mV with 10 μm RR; n = 4), suggesting that Mg2+ and RR compete with each other for one or more binding sites within the channel pore. Alternatively, Mg2+ might have an allosteric effect on the RR binding site. TRPM6-induced currents increased significantly upon dialysis with Mg2+-free intracellular solutions (e.g. Fig. 2a), indicating that intracellular Mg2+ has an inhibitory effect on TRPM6 activity. To test the effect of intracellular Mg2+ in a more direct manner, we performed experiments in which the intracellular Mg2+ concentration was directly altered in a spatially uniform manner using flash photolysis of the photolabile Mg2+chelator DM-nitrophen (19.Kaplan J.H. Ellis-Davies G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6571-6575Crossref PubMed Scopus (258) Google Scholar). Fig. 4a shows an experiment in which a TRPM6-expressing cell was intracellularly dialyzed with a pipette solution containing 5 mm DM-nitrophen 90% saturated with Mg2+ as well as the Mg2+-sensitive dye Magfura-2. After maximal activation of the current, a brief flash (∼1 ms) of UV light was applied to the cell and the tip of the patch pipette, which resulted in a stepwise increase of the intracellular Mg2+ concentration from < 0.1 mm to ∼0.5 mm (Fig. 4a, top). As a result of the increased intracellular Mg2+, the TRPM6-induced current was significantly reduced, both at positive and negative potentials (Fig. 4a, bottom). We observed two temporal phases in the Mg2+-induced current reduction. As shown in Fig. 4b, the outward current at +100 mV rapidly decreased after the flash-induced increase in intracellular Mg2+ with an initial time constant of 24 ± 5 ms at 0.51 ± 0.07 mm Mg2+ (n = 5). Thereafter, a second, slower phase of inhibition was observed, which was complete in ∼ 30–40 s (time constant 21 ± 3 s; n = 5). Fig. 4c compares current-voltage relations obtained just before and, respectively, 5 and 45 s after the flash, illustrating that the inhibitory effect of intracellular Mg2+ displays little or no voltage dependence. Fig. 4d displays the relative current 45 s after the flash in the function of the post-flash Mg2+ concentration. The solid line (Fig. 4d) represents a dose-response curve fitted to the data, which yielded an apparent KD of 0.51 mm and a Hill coefficient of 2.1. Most of our experiments were performed with a pipette solution containing 10 mm EDTA, which causes slow passive depletion of the intracellular Ca2+ stores. Given that several members of the TRP family have been shown to be store-dependent (4.Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), we reasoned that part of the current activation after establishment of the whole-cell configuration might reflect store dependence of TRPM6. To test this possibility, we measured whole-cell currents using a Mg2+-free pipette solution containing 0.1 mm EGTA as the sole Ca2+ buffer, which is not sufficient to cause significant store depletion within the time course of our experiments. Under this condition, TRPM6-expressing cells still developed robust outwardly rectifying currents, and the average current density (319 ± 58 pA/pF; n = 8) was not different from that of cells dialyzed with the standard solution containing 10 mm EDTA (341 ± 24 pA/pF; n = 42; see Fig. 1b). Moreover, depletion of the intracellular stores by extracellular application of the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor 2,5-di-t-butyl-1,4-benzohydroquinone (20 μm) or the Ca2+ ionophore ionomycin (2 μm) did not alter the amplitude of the TRPM6-induced currents measured with 0.1 mm EGTA in the pipette solution (n = 3 for each drug; data not shown). From this we conclude that the activity of TRPM6 does not critically depend on the filling state of the intracellular Ca2+ stores. The TRPM6-induced currents are highly reminiscent of the currents induced by overexpression of TRPM7 in HEK-293 or Chinese hamster ovary cells (5.Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar, 13.Nadler 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) as well as to the outwardly rectifying currents regulated by intracellular Mg2+ and/or Mg2+-nucleotides that are present in cell types (including HEK-293 cells) that endogenously express TRPM7 and have been termed MagNuM (for Mg2+-nucleotide-inhibited) or MIC (Mg2+-inhibited cation) currents (13.Nadler 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, 20.Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (267) Google Scholar, 21.Hermosura M.C. Monteilh-Zoller M.K. Scharenberg A.M. Penner R. Fleig A. J. Physiol. 2002; 539: 445-458Crossref PubMed Scopus (165) Google Scholar, 22.Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar). The list of similarities between the TRPM6-induced currents and the TRPM7, MagNuM, or MIC currents includes the outwardly rectifying current-voltage relation, the lack of voltage dependence, and the sensitivity to intracellular Mg2+. This raises the important question of whether the currents described in this study are mediated by TRPM6 itself or rather represent the activity of the endogenously expressed TRPM7. However, this possibility is rather unlikely for the following reasons. First, current amplitudes in TRPM6-expressing cells were ∼10 times larger than in non-transfected cells, and developed much faster when intracellular Mg2+ was buffered. Second, no significant increase in outwardly rectifying currents was observed upon overexpression of two HSH-causing TRPM6 mutants. Finally, the average current density in TRPM6-expressing HEK-293 cells measured in this study was of the same order of magnitude and even larger than those reported for HEK-293 or CHO cells overexpressing TRPM7 under comparable experimental conditions (5.Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (625) Google Scholar, 13.Nadler 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, 20.Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (267) Google Scholar, 21.Hermosura M.C. Monteilh-Zoller M.K. Scharenberg A.M. Penner R. Fleig A. J. Physiol. 2002; 539: 445-458Crossref PubMed Scopus (165) Google Scholar, 22.Kozak J.A. Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 2002; 120: 221-235Crossref PubMed Scopus (169) Google Scholar, 23.Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (460) Google Scholar). Thus, the most straightforward explanation of our findings is that TRPM6 forms all or a major part of a Mg2+-permeable cation channel regulated by intracellular Mg2+. TRPM6 and TRPM7 have an overall amino acid sequence identity of 52%, which increases to >80% in the region between the fifth and sixth transmembrane region where the pore-forming loop is presumably located. Moreover, both TRPM6 and TRPM7 have a carboxyl-terminal region with sequence similarities to the atypical α-kinase family. Given this high degree of homology, it cannot be excluded that TRPM6 and TRPM7 are able to form heterotetrameric channels, similar to what has been described for TRPV5/TRPV6 (24.Hoenderop J.G. Voets T. Hoefs S. Weidema A.F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (295) Google Scholar) and TRPC1/TRPC5 (25.Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). However, the 10-fold larger current amplitude in the TRPM6-expressing cells compared with non-transfected HEK-293 cells indicates that the majority of the channels are actually TRPM6 homotetramers. As a consequence of our findings, some caution is warranted when equating endogenous MagNuM (MIC) currents to TRPM7, as TRPM6 or putative TRPM6-TRPM7 heteromultimers might underlie these currents in some cell types. In conclusion, we have demonstrated that TRPM6 confines a Mg2+-permeable channel that is specifically localized to the apical membrane of Mg2+-reabsorbing tubules in the kidney and the brush-border membrane of the Mg2+ absorptive cells in the duodenum. The tight regulation of the TRPM6-induced current by intracellular Mg2+ provides a feedback mechanism on Mg2+ influx and implies that intracellular Mg2+ buffering and Mg2+ extrusion mechanisms strongly impact on channel functioning. Parvalbumin and calbindin-D28K, which, as shown in this work, are co-expressed with TRPM6, might function as intracellular Mg2+ chelators in the DCT, although further studies are needed to substantiate this. To our knowledge, TRPM6 represents the first molecularly identified and functionally characterized component of active Mg2+ (re)absorption. The absence of the TRPM6-dependent Mg2+ influx pathway in kidney and the small intestine provides a straightforward explanation for the severe hypomagnesemia observed in HSH patients. We thank Drs. A. Ryazanov for kindly providing a partial cDNA clone encoding human TRPM6 (GenBank™ accession number AF350881), J. Loffing for the antibody against NCC, J. Prenen and A. Janssens for technical assistance, and K. Talavera, G. Owsianik, and C. van Os for stimulating discussions.
Referência(s)