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J. Biol. Chem., Vol. 280, Issue 21, 20793-20803, May 27, 2005
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¶
From the
First Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan, the Department of Structure Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma Nara 630-0101, Japan, ||Laboratory of Molecular and Cellular Neuroscience and **Laboratory of Mass Spectrometry, The Rockefeller University, New York, New York 10021, the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4561, and the ¶¶Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508
Received for publication, December 6, 2004 , and in revised form, February 22, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1 as bait (and termed TRP-PLIK) (5) and in a screen for TRP-related channels (and termed LTRPC7) (6). Based on revised nomenclature for TRP channels, the kinase-containing TRP has been termed TRPM7/ChaK1 based on its similarity to long TRP family members, melastatin, MTR1 (TRPM5), and LTRPC2 (TRPM2) (7). A closely related gene, TRPM6/ChaK2 has also been identified as being mutated in familial hypomagnesemia (8, 9), and a key role for both TRPM6 and TRPM7 has been suggested in Mg2+ homeostasis (8-11).
Members of the TRP family of channels contain six predicted transmembrane segments and are related to the superfamily of ion channels that include voltage-gated K+ channels and cyclic nucleotide-gated channels (12, 13). In addition to the kinase domain at the C terminus of TRPM7, other regions of potential functional importance are a putative coiled-coil domain and a domain enriched in Ser/Pro and Thr/Pro motifs (the STP domain) (Fig. 1A). We have recently determined the crystal structure of the kinase domain of TRPM7 (4). The TRPM7 kinase domain (residues 1551-1577) forms a domain-swapped bilobate dimer. Despite a lack of amino acid sequence similarity to the classical eukaryotic protein kinases, the TRPM7 kinase domain exhibits within its N-terminal lobe secondary and tertiary structural similarity to the protein kinase A superfamily as well as to metabolic enzymes with ATP grasp domains. However, in contrast to the classical kinase superfamily, the C-terminal lobe of the TRPM7 kinase domain contains a zinc finger homology domain, and zinc appears to play a structural role in TRPM7 and related EF2K/MHCK family members (4).
Previous studies in heterologous expression systems have indicated that TRPM7 exhibits a steep outwardly rectifying current-voltage relation and permeability to monovalent and divalent cations (5, 6). Nadler et al. (6) demonstrated that millimolar concentrations of internal Mg2+, MgATP, MgGTP, or other Mg2+-nucleotides inhibit TRPM7. In RBL and Jurkat T cells, the native correlate of TRPM7 was described and termed MagNuM (magnesium nucleotide-dependent metal cation) (14), or MIC (Mg2+-inhibited cation) (15, 16). Similar currents have also been described in cardiac fibroblasts (17), smooth muscle cells (18), and brain microglia (19).
The role of the kinase domain and phosphorylation in the ion channel function is controversial (for a recent review, see Ref. 20). Runnels et al. (5) reported that mutations of kinase domain residues (probably involved in ATP and zinc binding (4)) apparently abolished channel activity measured in whole-cell recording. Subsequently, Schmitz et al. (10) showed that whereas TRPM7 mutants with impaired kinase activity formed functional channels, these channels exhibited reduced sensitivity to Mg2+. Moreover, a kinase deletion mutant exhibited increased Mg2+ sensitivity. From these studies, Schmitz et al. (10) concluded that the TRPM7 kinase domain was somehow coupled negatively to the mechanism(s) that mediated Mg2+-dependent inhibition.
In this study, we have characterized the biochemical and functional properties of the TRPM7 kinase activity. Guided by the crystal structure, we have confirmed the identity of amino acids critical for kinase activity. We have identified two major sites of TRPM7 autophosphorylation by mass spectrometry and demonstrated that these sites are phosphorylated in intact cells. Using whole-cell patch clamp recording, we have measured the magnitude of the constitutive current conducted by wild-type TRPM7 and its kinase-dead and autophosphorylation site variants and compared the extent of inhibition by internal Mg2+ among them. We have also measured the corresponding Ca2+ influx in intact mammalian cells overexpressing the wild-type channel and the variants. We show that autophosphorylation site or kinase-dead TRPM7 mutant channels do not differ from wild-type channels in their ability to form functional ion channels and in their sensitivity to internal Mg2+. Thus, TRPM7 channel function and regulation by Mg2+ are clearly dissociated from autophosphorylation or kinase activity. Interestingly, results obtained following deletion of the much of the kinase domain suggest that this C-terminal region may play a structural role in channel assembly or subcellular localization.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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KD, and TRPM7 mutants were cloned into the pcDNA3.1 vector (Invitrogen). Expression of Wild-type and Mutant TRPM7 Kinase Domain in Sf9 Cells—A recombinant 316-residue kinase domain fragment (TRPM7 residues 1548-1863) was expressed in Sf9 insect cells with a hexahistidine tag by using the Bac-to-Bac baculovirus expression system (Invitrogen) essentially as described (4). The expressed protein was purified using Q-Sepharose (Amersham Biosciences) and Ni2+-nitrilotriacetic acid Superflow (Qiagen, Hilden, Germany). A PCR-based site-directed mutagenesis method was employed to generate point mutants, R1622L, K1646R, K1727A, N1731V, D1765N, D1765A, Q1767N, Q1767A, T1774S, T1774A, D1775A, and N1795A. The wild type pFastBac construct was used as a template, and PCR was carried out for each mutant with a set of sense and antisense mutagenic primers (essentially as described in the Stratagene QuikChangeTM kit). The PCR products were treated with DpnI restriction enzyme and purified through agarose gel electrophoresis. Purified DNA was used to transform Escherichia coli (DH5a), and transformants harboring mutant constructs were identified. Mutations were confirmed by DNA sequencing. Mutant pFastBac constructs were used to generate mutant recombinant viruses, and protein was expressed and purified as in the case of wild type.
Expression of GST-TRPM7 Kinase Domain in E. coli—For bacterial expression, DNA fragments were subcloned in frame with GST using the pGEX-4T-2 vector (Amersham Biosciences). GST fusion proteins including either residues 1180-1863 or 1580-1863 of TRPM7 were expressed in E. coli (DE3) and purified using standard methods.
Phosphorylation Assays—Reaction mixtures (100 µl) contained 50 mM HEPES (pH 7.5), 10 mM magnesium acetate (unless otherwise indicated), 5 mM dithiothreitol, 100 µM [32P]ATP (specific activity, 2-5 x 102 cpm/pmol), and GST-TRPM7-KD (10 µg/ml) or Sf9 cell TRPM7-KD or mutant proteins (
1 µg/ml), with or without myelin basic protein (MBP; 50 µg/ml). For analysis of the effect of Mg2+ and other divalents cations on kinase activity, GST-TRPM7 was incubated with MBP in a reaction mixture containing different concentrations of Mg2+, Ca2+, and Zn2+. All reactions were initiated by the addition of [32P]ATP and carried out at 30 °C for 10-30 min. In the studies shown in Figs. 3D and 4B, GST-TRPM7-(1180-1863) (wild-type sequence), GST-TRPM7-(1180-1863) (S1511A and S1567A; see details below), and GST-TRPM7-(1180-1863) (TAP sequence; see details below) (100 ng/ml) were incubated in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 20 mM EGTA, 1 mM dithiothreitol, 100 µM ATP for 30 min at 30 °C (in Fig. 3D, [32P]ATP was also included). Reactions were terminated by the addition of SDS sample buffer, and samples were analyzed by SDS-PAGE (10% polyacrylamide). Gels were stained with Coomassie Brilliant Blue, destained, dried and subjected to autoradiography or analysis using a phosphor imager (Fuji). Phosphoamino acid analysis and two-dimensional peptide mapping were performed as described (21).
Phosphorylation in Intact Cells—HEK293 cells were transfected with wild-type TRPM7 using FuGENE 6 (Roche Applied Science). Wild-type TRPM7 was expressed for 24-48 h, and cells were labeled with 32Pi, for 2 h. Cells were incubated without or with ionomycin (5 µM) for 5 min, cell lysates were prepared, and TRPM7 was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma). Samples were analyzed by SDS-PAGE and autoradiography. In other studies, HEK293 cells were transfected with wild-type TRPM7 or TRPM7-TAP using Lipofectamine 2000 (Invitrogen). Cell lysates were prepared, and TRPM7 was immunoprecipitated with anti-FLAG M2 affinity gel. Samples were analyzed by SDS-PAGE and immunoblotted using phospho-specific or FLAG antibodies.
Mass Spectrometry—After SDS-PAGE, bands containing autophosphorylated TRPM7 were excised from the gel, destained, and digested with trypsin (Roche Applied Science). The tryptic peptides were eluted from the gel and collected on POROS R2 beads (Applied Biosystems, Foster City, CA). After washing, the peptides were eluted from the beads and spotted onto a matrix-assisted laser desorption/ionization (MALDI) target using 2,5-dihydrobenzoic acid (Sigma) as matrix. MALDI time-of-flight (TOF) mass spectrometry was carried out on a Voyager-DE STR instrument (Applied Biosystems, Foster City, CA). MALDI-ion trap analysis was performed on a modified LCQ (Thermo Finnegan, San Jose, CA) mass spectrometer (22). On-line HPLC-MS/MS was performed using a 0.2 x 50-mm MAGIC MS C18 column (Michrom Bioresources, Auburn, CA) connected directly to an LCQ-DECA electrospray-ion trap mass spectrometer (Thermo Finnegan).
Immunofluorescence and Immunoblotting—Antibodies to TRPM7 were made against GST fusion proteins that contained the coiled-coil domain (residues 1145-1321) (in rabbit) or the kinase domain (residues 1580-1863) (in guinea pig) of TRPM7. A phosphorylation state-specific antibody that specifically recognizes phospho-Ser1567 of TRPM7 was generated in rabbit using as antigen a phosphopeptide corresponding to residues 1562-1571 of mouse TRPM7. Antibodies were affinity-purified by absorption to immobilized recombinant proteins or phosphopeptide. For indirect immunofluorescence using confocal microscopy, HEK293 cells were fixed with 4% formaldehyde in PBS and permeabilized in 0.1% Triton X-100 (in PBS). After blocking (5% milk powder) for 30 min, cells were incubated with anti-TRPM7 antibody (guinea pig) for 2 h, washed three times in washing buffer, and incubated with fluorescein isothiocyanate-conjugated anti-guinea pig antibody for 30 min. Immunoblotting was carried out using standard procedures, and proteins were identified by ECL (Amersham Biosciences).
Ca2+ Imaging of HEK293 Cells—HEK293 cells (RIKEN, Tsukuba, Japan) were transfected with wild-type TRPM7 and various mutants using Lipofectamine 2000 reagent (Invitrogen). Transfection efficiencies, estimated by immunofluorescence staining, were typically >80%. Transfected cells were washed in HEPES-buffered saline (120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, 11.1 mM glucose, 20 mM HEPES-Na (pH 7.4)) and loaded with 5 µM FURA-2/AM (Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Cells were then incubated in HEPES-buffered saline for 20 min at 37 °C to allow cleavage of the acetoxymethyl ester. Fluorescence images of the cells were recorded at room temperature and analyzed with a video image analysis system (AquaCosmos; Hamamatsu Photonics, Hamamatsu, Japan), with excitation alternatively at 340 and 380 nm and emission at 510 nm. Data from 30-50 individual cells were collected per experiment, and ensemble averages were calculated from multiple experiments. Data acquisition was typically for 5-s intervals and lasted for 15 min.
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12 h. For mock transfection, the same procedure was employed, except DNA was omitted. Cells were subsequently trypsinized and plated on acid-washed glass coverslips for recording. For whole-cell patch clamp recording, coverslips with CHO cells were placed on a stage of an inverted microscope (Zeiss IM-35) and superfused with the external solution used for recording. GFP-positive cells were visualized using mercury arc lamp illumination at 290-nm wavelength. Recombinant TRPM7 channel currents were recorded in the whole-cell patch clamp using an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Command voltage ramps from -110 to +85 mV (211-ms duration) were applied at 0.5-Hz frequency, and the resulting currents were digitized at 200 µs. Cell and electrode capacitance was compensated with the EPC-9 circuitry. Series resistance was compensated by 85% to avoid potential errors due to large current amplitudes. Resistances of the fire-polished pipettes measured with the internal solutions were 1.5-4 megaohms. The silver-silver chloride ground electrode was connected to the bath solution via an agar bridge. All recordings were made at room temperature. The current amplitude was measured at +80 mV and plotted against time after whole cell recording mode was established. In the majority of cells, the current had reached its maximum less than 6 min after break-in. All CHO cells tested exhibited an endogenous MIC current thought to represent native TRPM7 channels. The endogenous MIC current amplitude at +80 mV averaged 30.6 pA/picofarad (S.E. = 5.6, n = 12). Igor Pro (Wavemetrics, Lake Oswego, OR) and Microcal Origin (Microcal Software, Northampton, MA) were used to analyze the data. The following internal solutions were used to record from CHO cells overexpressing wild-type TRPM7 and mutants. The 0 Mg2+ solution contained 130 mM Cs+ glutamate, 12 mM EGTA, 0.9 mM CaCl2, 10 mM HEPES, pH 7.3. The 4 and 6.5 mM Mg2+ solutions had 130 mM Cs+ glutamate, 12 mM EGTA, 0.9 mM CaCl2, 4 mM/6.5 mM MgCl2, 10 mM HEPES, pH 7.3. The free Mg2+ concentrations in these solutions were 2.3 and 4 mM, respectively, as calculated by Maxchelator software. The external solution contained 4.5 mM K, 162 mM Na+ aspartate, 2 mM CaCl2, 10 mM HEPES, 2 mM glucose, pH 7.3. For testing Ca2+ and Zn2+ inhibition of TRPM7 current, EGTA in the internal solution was replaced with 1 mM EDTA and 5 mM CaCl2 or ZnCl2 added as indicated. Salts were purchased from Sigma and Calbiochem.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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We recently determined the crystal structure of the kinase domain of TRPM7 (4). Whereas the likely function of several key amino acids in the kinase domain of TRPM7 was implied by analogy with residues in the classical kinase superfamily, it was important to directly analyze the properties of these residues using site-directed mutagenesis. Moreover, the availability of mutant (kinase-dead) TRPM7 was necessary for the studies described below. Mutation of residues (Arg1622 to leucine; Lys1727 to alanine; Asn1731 to valine; Thr1774 to serine or alanine) likely to be involved directly in binding of phosphate groups of ATP or residues (Gln1767 to asparagine or alanine; Asp1775 to alanine) likely to be involved in metal binding all resulted in very low activity (Fig. 2, A and B). Similarly, mutation of residues implicated in catalysis (Lys1646 to arginine; equivalent to the conserved lysine residue of classical kinases that is often mutated to produce a kinase-dead protein; Asp1765 to asparagine or alanine) resulted in very low activity. Mutation of a residue in the glycine motif (Asn1795 to alanine) that may be involved in peptide substrate binding or orientation of the substrate-binding loop resulted in low but measurable activity.
Mass Spectrometric Analysis of Phosphorylation Sites in TRPM7—TRPM7-(1180-1863) phosphorylated in vitro as described above was subjected to SDS-PAGE. The TRPM7 band was digested in the gel with trypsin, and the tryptic peptides were analyzed by MALDI-TOF mass spectrometry (Fig. 3A). Since the addition of HPO3 causes an increase in mass of 80 Da, potential phosphopeptides within the tryptic map were identified by searching the observed signals for shifts of 80 Da relative to expected tryptic peptides. Three peptides showing an 80-Da shift were observed, corresponding to residues 1564-1576 (predicted phosphorylated mass 1516.7 Da, observed mass 1516.4 Da), 1501-1521 (predicted mass 2380.5, observed mass 2380.3), and 1564-1584 (predicted mass 2418.7, observed mass 2418.4). These peptides were subjected to fragmentation in a MALDI-ion trap mass spectrometer (22); each one showed a facile loss of 98 Da due to elimination of the elements of H3PO4, confirming that each was phosphorylated. Other fragment ions confirmed the identity of each tryptic peptide. The MS2 spectrum of the 1501-1521 peptide contained fragment ions showing that the major site of phosphorylation in this peptide was Ser1511 (data not shown). The MALDI-ion trap fragmentation data from the other two peptides did not allow us to determine whether Ser1565 or Ser1567 was the site of phosphorylation, so the 1564-1584 peptide was analyzed by HPLC-MS/MS (Fig. 3, B and C). The fragmentation pattern of the triply charged ion indicated that Ser1567 was phosphorylated in the 1564-1584 peptide.
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Phosphorylation of TRPM7 in Intact Cells—To investigate whether Ser1511 and/or Ser1567 were phosphorylated in intact cells, wild-type TRPM7 was expressed in HEK293 cells, cells were labeled with 32Pi, and the phosphorylation of TRPM7 analyzed following immunoprecipitation (Fig. 4A). In control conditions, TRPM7 was strongly labeled with phosphorylation occurring almost exclusively on serine residues (Fig. 4E). Comparison of the phosphopeptide tryptic map of full-length TRPM7 phosphorylated in HEK293 cells (Fig. 4C) with that of TRPM7-(1180-1863) autophosphorylated in vitro (see Fig. 1E for comparison) indicated that the major phosphorylated sites were included within residues 1180-1863 of TRPM7. The phosphorylation of Ser1567 in intact cells was confirmed using the phospho-Ser1567 antibody (Fig. 4C). Notably, no phosphorylation of Ser1567 was found in the inactive TRPM7-TAP mutant. Together, these studies of phosphorylation of TRPM7 in vitro and in intact cells indicate that Ser1511 and Ser1567 are the major autophosphorylation sites in TRPM7, and at least under basal conditions TRPM7 is not likely to be phosphorylated by other protein kinases in intact cells.
Characterization of the Role of Autophosphorylation and Kinase Activity of TRPM7 in Regulation of Ca2+ Influx—To examine the effects of the kinase activity of TRPM7 on its functional properties, HEK293 cells were transiently transfected with DNA for wild-type or mutant TRPM7. In initial experiments with wild-type TRPM7 and using an antibody raised against the kinase domain, TRPM7 was found to be expressed in a high percentage of cells (>80%), where it was largely localized in the plasma membrane, although some punctate intracellular immunofluorescence was observed (Fig. 5A). No immunofluorescence was observed in cells transfected with the empty vector. By immunoblotting with an antibody to the coiled-coil domain, full-length TRPM7 was detected as a protein of 210 kDa, a molecular mass consistent with that predicted from the cDNA sequence (212 kDa) (see Fig. 6A).
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We further assessed the potential role of the kinase domain of TRPM7 by expressing a mutant (TRPM7-(1-1599)) in which most of the kinase domain was deleted (Fig. 6). The expression levels of wild-type TRPM7 and TRPM7-(1-1599) (KD) in HEK293 cells were comparable as measured by immunoblotting using an anti-coiled-coil domain antibody (Fig. 6A). In contrast to the full-length kinase-dead TRPM7 (see Fig. 5D), very little Ca2+ influx was observed in cells expressing TRPM7-KD (Fig. 6B). A similar result was observed using a different protocol in which cells were incubated initially with Ca2+-free buffer (0.5 mM EGTA) and thapsigargin (200 nM) to deplete intracellular stores, and then Ca2+ influx was measured upon the addition of 2 mM CaCl2 to the extracellular medium (Fig. 6C).
Whole-cell Patch Clamp Recordings from CHO Cells Overexpressing Wild-type and Mutant TRPM7 Proteins—Inclusion of millimolar internal Mg2+, MgATP, or MgGTP inhibits the development of TRPM7 current (6). Previous studies have demonstrated that wild-type TRPM7 channel activity can be induced during whole cell recording by lowering internal Mg2+ by dialysis with pipette solutions lacking Mg2+ (5, 6). We therefore evaluated channel activity by whole-cell recording using Mg2+-free internal solutions in CHO cells that expressed wild-type and mutant TRPM7. A typical I-V relationship (top) and time course for development of the wild-type TRPM7 current is shown (Fig. 7A). Immediately following break-in to achieve whole-cell recording, the current was small but detectable (20-s trace) and increased gradually over the course of several minutes (6-min trace). The autophosphorylation site mutant (Fig. 7, B and E) and the kinase-dead mutant (Fig. 7, C and E) also exhibited currents that developed with I-V characteristics and a time course which was the same as wild-type TRPM7. The current immediately after break-in represents activity of preactivated channels and was variable from cell to cell (shown in Fig. 7E). Moreover, the kinetics of the current development and rundown varied greatly from cell to cell but did not depend on the expressed construct (compare Fig. 7, A-C). In contrast, the TRPM7-KD mutant failed to express significant channel activity (Fig. 7, D and E). The comparatively small current in untransfected CHO cells is the endogenous MIC current, which is thought to represent native TRPM7 protein (Fig. 7D, and data not shown for mock-transfected cells). Thus, although deletion of the kinase domain abolished TRPM7 channel activity, the endogenous current was unaffected.
We next examined Mg2+ inhibition of wild-type TRPM7 and the various mutants by comparing the extent of inhibition by two concentrations of internal Mg2+ (2.3 and 4 mM free concentrations) (Fig. 8 and data not shown). Preactivated current was detected immediately after break-in, and the current subsequently declined over minutes as Mg2+ entered the cell (Fig. 8, A-D, shows representative results for 2.3 mM free Mg2+). No apparent difference was detected between the time courses of Mg2+ inhibition between wild type, the double autophosphorylation site, or the kinase-dead mutant. Normalized current amplitudes at break-in for individual cells are summarized in Fig. 8E. In order to address the extent of Mg2+ inhibition quantitatively, we measured the current amplitude after steady-state inhibition was established (usually 400 s after break-in) and divided this value by the amplitude at break-in (I0). This approach was chosen because channel expression varied greatly from cell to cell (Fig. 8E), and it allowed us to measure relative Mg2+ inhibition for each cell individually. The extent of Mg2+ inhibition determined in this fashion was not significantly different at either 2.3 or 4 mM Mg2+ for wild-type TRPM7 and the autophosphorylation site or kinase-dead mutants (Fig. 8F).
Effects of Divalent Cations on the Kinase and Channel Activities of TRPM7—In further experiments, we compared the effects of divalent ions on both channel and kinase activities of TRPM7. As expected, MBP kinase activity required Mg2+ with maximal activity being observed with a concentration of 10 mM (Fig. 9A). In the presence of 10 mM Mg2+, the addition of Ca2+ had no effect on kinase activity (Fig. 9A) or autophosphorylation (data not shown). Notably, the addition of Ca2+ plus calmodulin resulted in inhibition of MBP phosphorylation, presumably as a result of binding to the MBP substrate (24). In the presence of 10 mM Mg2+, the addition of high concentrations of Zn2+ strongly inhibited kinase activity (Fig. 9A) and autophosphorylation (data not shown), presumably by competing with Mg2+. Similar results were observed in a recent study by Ryazanov et al. (24) (see also Schmitz et al., (10)).
We next examined effects of Ca2+ and Zn2+ on TRPM7 current. In a control experiment where the internal solution contained no divalent cations (1 mM EDTA), TRPM7 current increased over time (Fig. 9B). The addition of 5 mM CaCl2 (Fig. 9C) or 5 mM ZnCl2 (Fig. 9D) reduced TRPM7 current markedly (note different y axis units). The extent of inhibition was similar to that of Mg2+ (see Fig. 8) in that it was voltage-independent and proceeded with a similar time course. Thus, although Mg2+ is required, Ca2+ has no effect, and Zn2+ inhibits TRPM7 kinase activity, all three cations inhibited TRPM7 channel current.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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- and 
-phosphates of ATP (4).
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helix (A) of the kinase domain, the A helix being the main structural element of the segment that is "exchanged" between the two monomers. The autophosphorylation of these sites appears to have no effect on kinase activity (24), but it seems possible that phosphorylation of Ser1567 in particular has the potential to affect the dimeric structure or stability of the paired kinase domains.
Previous studies have shown that EF2 kinase and MHCK both phosphorylate threonine residues within their respective substrates (25, 26). These results raised the possibility that atypical kinases might be selective for threonine. However, the two autophosphorylation sites identified in this study are serine residues, and serine is the residue that is phosphorylated in MBP. The EF2K/MHCK family of atypical protein kinases are unlikely, therefore, as a group to show preference for threonine in their respective substrates. Moreover, the EF2K/MHCK family kinases do not seem to recognize any obvious consensus phosphorylation site motif. Ser1511 and Ser1567 are found within the amino acid sequences, STEDSPEVDS(P)KAALLP-DWLR and MRLSQS(P)IPFVPVPPR, respectively (boldface type indicates phosphorylated serines). These sites do not exhibit any similarity to each other or to sites phosphorylated by other EF2K/MHCK family members. Structure-function studies of EF2 kinase and MHCK have suggested that interactions with substrate require additional protein/protein interactions outside of the immediate contacts at the active site. For example, EF2 interacts directly with the extreme C terminus of EF2 kinase, a region removed from the catalytic domain by 300 amino acids (25, 27, 28). In MHCK-A, the catalytic domain is flanked at the N terminus by a coiled-coil region, and at the C terminus by a 7-fold WD repeat motif. Removal of the WD repeat domain decreased significantly the rate of phosphorylation of full-length myosin but had no effect on the kinetics of phosphorylation of a short synthetic peptide that served as a substrate for the kinase (29). Presumably, these additional targeting interactions stabilize the interaction of EF2 or myosin with their respective kinase and perhaps orient the region containing the phosphorylation sites of EF2 or myosin in the correct position in the active sites of either kinase. Similar secondary interaction domains away from the active site of the kinase may also be required for recognition of substrates for TRPM7.
Our studies indicate that the major sites autophosphorylated in vitro, Ser1511 and Ser1567, are also the main sites phosphorylated in intact cells, at least under control conditions. It is possible that upon stimulation, one or more kinases could phosphorylate TRPM7 and regulate kinase or channel function. It is also possible that Ca2+ or other cations that permeate the TRPM7 channel pore could have a local effect on kinase function.
Does the TRPM7 Kinase Domain Play Any Role in Regulation of Its Channel Function?—Given the unique incorporation of a kinase domain within the TRPM6 and TRPM7 polypeptides, it might have been expected that the kinase activity would play a significant role in channel gating. However, the results of the current study failed to find any obvious regulatory function for the TRPM7 kinase activity. We assessed channel function in several different ways and analyzed the effect of several different types of mutations of TRPM7. Measurement of Ca2+ influx into intact cells, or by carrying out whole-cell recording with Mg2+-free internal solutions, indicated that mutation of the major autophosphorylation sites in TRPM7 or inactivation of kinase catalytic function had no significant effect compared with results obtained with wild-type TRPM7. Our analysis of autophosphorylation site mutants has the added advantage of ruling out the possibility that low levels of endogenous TRPM7 kinase activity might be sufficient to result in significant phosphorylation of exogenous TRPM7 kinase mutants.
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Schmitz et al. (10) also examined the sensitivity of kinase mutant channels to internal Mg2+ and showed that at lower concentrations (3 mM) Mg2+, inhibition was reduced for the two kinase domain mutants, whereas at higher concentrations (7-8 mM), inhibition was identical to the wild-type channel. This differs from our results indicating that TRPM7 kinase activity had no effect on Mg2+ inhibition. The reason for this discrepancy is unknown at present. It is worth mentioning, however, that Schmitz et al. (10) measured averages of current amplitudes for groups of cells, whereas we compared TRPM7 inhibition in individual cells and averaged those. It is possible that variability of channel expression influenced the analysis of Schmitz et al. (10).
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KD deletion mutant was similar to that of wild-type TRPM7. However, we cannot rule out the possibility that the protein was not normally assembled as a tetramer and/or processed and trafficked to the plasma membrane. Schmitz et al. (10) also examined the properties of a slightly shorter TRPM7-KD deletion mutant (residues 1-1569). Whole-cell recordings indicated that cells expressing the 1-1569 TRPM-KD deletion mutant exhibited significantly reduced TRPM7 currents (10% of that of cells expressing wild-type TRPM7) perhaps reflecting different properties of the mutant used or of its expression level. Schmitz et al. (10) also suggested that their TRPM7-KD deletion mutant was much more sensitive to inhibition by Mg2+. However, given the much reduced currents, it is difficult to derive clear conclusions from the data shown. We also examined the regulation of channel function and kinase activity by different divalent cations. The results from measurement of kinase activity in vitro indicated that while Mg2+ is required for kinase activity, Ca2+ has no effect, and Zn2+ inhibits kinase activity. These results are in complete agreement with a recent study by Ryazanova et al. (24). However, in circumstantial support of the conclusion that kinase activity is dissociated from channel function, these three cations inhibited TRPM7 channel current (see also Ref. 30). Additional circumstantial evidence for lack of role of kinase activity in regulation of channel function comes from consideration of the fact that both MgATP and MgGTP inhibit channel activity (6), whereas only MgATP is capable of supporting TRPM7 kinase activity (24).
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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FOOTNOTES |
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¶ Recipient of an American Heart Association Postdoctoral Fellowship.
Recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.
|||| To whom correspondence should be addressed: Dept. of Psychiatry, Yale University School of Medicine, New Haven, CT 06508. Tel.: 203-974-7725; Fax: 203-974-7724; E-mail: angus.nairn{at}yale.edu.
1 The abbreviations used are: EF2K, eukaryotic elongation factor-2 kinase; MHCK, myosin heavy chain kinase; MBP, myelin basic protein; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; HPLC, high pressure liquid chromatography; MS, mass spectrometry; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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