From the
Metabolic and Cardiovascular Diseases Drug Discovery, ¶Applied Genomics, and ||Discovery Toxicology, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, New Jersey 08543
Received for publication, August 9, 2002 , and in revised form, November 4, 2002.
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ABSTRACT |
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INTRODUCTION |
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Following the identification of the founding member of this family, dTRP, which is from a Drosophila mutant with abnormal visual signal transduction (2), mammalian homologues have been cloned and all of them contain a six-transmembrane domain followed by a TRP motif (XWKFXR). Based on homology, they are divided into three subfamilies: TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) (3). Members of the TRPM subfamily have unusually long cytoplasmic tails at both ends of the channel domain, and some of the family members have an enzyme domain in the C-terminal region. Despite their similarities of structure, TRPMs have different ion-conductive properties, activation mechanisms, and putative biological functions. TRPM1 is down-regulated in metastatic melanomas (4). TRPM2 is a Ca2+-permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (5, 6), and changes in redox status (7). The TRPM2 gene is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holosencephaly (8). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca2+-permeable channel (9); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca2+ (10). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (11). TRPM7, another bifunctional protein, has kinase activity in addition to its ion channel activity. TRPM7 is regulated by Mg2+-ATP and/or inositol 1,4,5-disphosphate and is required for cell viability (12, 13, 14). TRPM8 is up-regulated in prostate cancer and other malignancies (15). Recently, it has been shown to be a receptor that senses cold stimuli (16, 17).
Using a bioinformatics approach, we have identified a member of the human TRPM subfamily that we have called hTRPM3, consistent with the unified TRP nomenclature (3). hTRPM3 contains long N and C termini, although it does not contain any additional enzymatic features. hTRPM3 mRNA is expressed primarily in kidney with lower levels in brain, testis, and spinal cord. When expressed in HEK 293 cells, hTRPM3 is co-localized with the plasma membrane and is capable of mediating Ca2+ entry. This hTRPM3-mediated Ca2+ conductance is partially lanthanide gadolinium (Gd3+)-sensitive and can be enhanced upon Ca2+ stores depletion or receptor activation.
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EXPERIMENTAL PROCEDURES |
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The phylogenetic tree for the TRPM subfamily was generated by the neighbor-joint method using the GCG GrowTree program with Kimura distance correction method. Members of TRP family with their respective GenBankTM accession numbers are as follows: hTRPM1 (NM_002420 [GenBank] ), hTRPM2 (NM_003307 [GenBank] ), hTRPM4 (NM_017636 [GenBank] ), hTRPM5 (NM_014555 [GenBank] ), hTRPM6,2 hTRPM7 (XM_030709), and hTRPM8 (NM_024080 [GenBank] ).
Cloning of hTRPM3Using the predicted exon genomic sequence from BAC AL358786 [GenBank] , primers were designed (forward: 5'-ATGTATGTGCGAGTATCTTTTGATACAAAACCT-3', 5'-GAAGGACACCAGGACATTGATTTG-3',5'-CAAGACCAGCCCTTCAGGAGTGAC-3', and 5'-CAGCTGGAAGACCTTATCGGGCG-3'; reverse: 5'-AGCCAAATCAATGTCCTGGTGTCC-3', 5'-GTCACTCCTGAAGGGCTGGTCTTG-3', 5'-CGCCCGATAAGGTCTTCCAGCTG-3', and 5'-TTAGGTGTGCTTGCTTTCAAAGCT-3') and used to amplify fragments from the human kidney Marathon Ready cDNA library (Clontech). The reaction mixture in 50 µl contains 5 µl of cDNA library, 0.5 mM each primer, 1.25 mM each dNTP, TaqPlus Precision buffer and 0.5 units of TaqPlus Precision polymerase buffer (Stratagene). The reaction was repeated for 30 cycles (94 °C for 45 s, 55 °C for 45 s, and 72 °C for 4 min). The amplified fragments were cloned into the sequencing vector pCR4 Blunt-TOPO (Invitrogen) for sequence analysis. The four fragments were then assembled to generate the full-length cDNA. For functional studies, the cDNA was fused in-frame with an HA epitope at its C terminus and subcloned into the mammalian expression vector pcDNA3.1/Hygro (Invitrogen).
Quantitative RT-PCRA PCR primer pair (forward: 5'-CGCAGCTGGAAGACCTTATC-3'; reverse: 5'-AAGCTGCTCTGACGGACAAT-3') was designed to measure the steady state levels of TRPM3 mRNA by SYBR Green real-time quantitative PCR using a standard protocol. All of the samples were run in triplicate.
The cDNA panel was made from mRNA purchased from Clontech. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair from cyclophilin. These data were used for normalization of the data obtained with the primer pair for the hTRPM3 transcript. The PCR data were converted into a relative assessment of the difference in transcript abundance among the tissues tested.
Northern Blot AnalysisHuman tissue Northern blots (Clontech) were probed with an RNA probe derived from a 645-bp DNA fragment amplified from the primer pair (forward: 5'-GAAGGACACCAGGACATTGATTTG-3'; reverse: 5'-AGGGAAGGGGAAGTGGTTGATCTC-3'). Hybridization of the blot was performed at 68 °C in ExpressHyb (Clontech) for 6 h with 1 x 106 cpm/ml 32P-labeled probe. Autoradiography was performed for 1 week at -70 °C.
In Situ HybridizationHuman Kidney was collected and received from the National Disease Research Interchange (Philadelphia, PA) according to Institutional Review Board-approved protocol. Tissue sections were embedded in Tissue Tek® O.C.T. compound (Sakura Finetek USA, Inc.) and snap-frozen by immersion in 2-methylbutane cooled in dry ice and subsequently stored at -70 °C. The sections were examined by a pathologist to ascertain the normality of the tissue before performing the following experiment.
Templates for hTRPM3 cRNA probes were derived from a 678-bp hTRPM3 fragment cloned in a pCR-BluntII-TOPO vector (Invitrogen) utilizing the primer pair: (forward: 5'-CAGCTGGAAGACCTTATCGGG-3'; reverse: 5'-TGGGAGGTGGGTGTAGTCTGAAGA-3'). The template for positive control cRNA human lysozyme probe was derived from a 638-bp cDNA expression sequence tag (Incyte Genomics) (GenBankTM accession number AA588081 [GenBank] ). 35S-Labeled riboprobes were synthesized via in vitro transcription utilizing the Riboprobe® Combination System (Promega) where T7 and Sp6 RNA polymerase yielded sense and antisense probes, respectively, for hTRPM3, whereas T7 and T3 RNA polymerases yielded antisense and sense probes, respectively, for human lysozyme. Cryostat tissue sections cut at 10 µm and fixed in 4.0% formalin were used for in situ hybridization as described previously (23). Tissue sections were acetylated, dehydrated in a graded ethanol series, immersed in chloroform, alcohol-rinsed, air-dried, and then hybridized with sense and antisense 35S-labeled RNA probes (1.5 x 106 cpm/slide) for 1620 h at 60 °C. Following hybridization, slides were rinsed in 4x SSC/50% formamide and 4x SSC, treated with RNase A (20 µg/ml, Invitrogen) at 37 °C, washed through increasing stringent solutions to a final high stringency wash in 0.1x SSC at 60 °C, dehydrated, air-dried, and then coated with NTB-2 emulsion (Eastman Kodak Co.). Slides were placed in a dark box with desiccant at 4 °C and developed after a 1- and 4-week exposure. Sections were stained with hematoxylin and eosin and coverslipped. Expression signals were detected by dark phase microscopy. Cellular phenotype identification was by bright field microscopy. The results have been confirmed in the kidney from three different donors and from nonhuman primate.
Mammalian Cell Expression and Immunofluorescence Staining of hTRPM3HEK 293 cells were cultured in Dulbecco's modified Eagle medium containing 10% heat-inactivated fetal bovine serum and grown on poly-D-lysine-coated glass coverslips. The cells were transiently transfected with hTRPM3-HA with FuGENE 6 (Roche Applied Sciences). 48 h later, cells were stained in culture media with the membrane probe Vybrant 228 CM-DiI (5µl/ml; Molecular Probes) at 37 °C for 5 min and 4 °C for 15 min. After washing with phosphate-buffered saline, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100, blocked in phosphate-buffered saline containing 5% fetal bovine serum and 5% normal goat serum, and stained with 10 µg/ml fluorescein-conjugated anti-HA high affinity antibody (3F10, Roche Applied Sciences) and 4',6-diamidino-2-phenylindole (0.5 µg/ml, Molecular Probes). Immuno-stained cell cultures were examined using a laser-scanning confocal microscope (ZEISS LSM510), a x63 oil immersion objective, and appropriate filter sets. Images shown are of a single optical section 1-µm thick.
Measurements of Changes in Intracellular Ca2+The cytoplasmic Ca2+ indicator Fluo-4-AM (Molecular Probes) and a fluorometric imaging plate reader (FLIPRTM, Molecular Devices) instrument were used to detect changes in intracellular Ca2+ concentration. The hTRPM3-transfected cells were seeded on poly-D-lysine-coated 96-well plates at a density of 70,000 cells/well 24 h after transfection and used 24 h after plating. Cells were loaded with 4 µM Fluo-4-AM at 37 °C for 30 min in a nominally Ca2+-free or 1 mM CaCl2 buffer containing (in mM): 140 NaCl, 4.7 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 2.5 Probenecid (Sigma), pH 7.4. Extracellular Fluo-4-AM was removed, and cells were maintained in either Ca2+-free buffer or buffer containing 1 mM Ca2+ at room temperature prior to the experiments, which were conducted within 30 min after dye removal. Fluo-4 was excited at 488 nm using an argon laser, and emitted light was selected using a 510570-nm bandpass filter. Base-line intracellular fluorescence was established during the initial 50 s of the FLIPR read, and then 1, 3, or 10 mM Ca2+ was added to each well and subsequent changes in the intracellular Ca2+ were monitored for 8 min. For store-depletion or receptor activation studies, 2 µM thapsigargin (TG) or 50 µM carbachol (CCh), respectively, was added to Fluo-4-loaded cells in Ca2+-free buffer before adding 2 mM Ca2+ on FLIPR. For pharmacology studies, 100 µM GdCl3 was added to Fluo-4-loaded cells in 0 or 1 mM Ca2+ buffer as described in figure legends prior to the start of the FLIPR recordings. Experiments were carried out at room temperature.
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RESULTS |
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hTRPM3 is predicted to be 1555 amino acids long and is comprised of the following characteristic features of a TRP channel: six transmembrane domains; an ion transport signature domain (amino acids 748959); a TRP signature motif (XWKFXR) located downstream of the sixth transmembrane region; and a coiled-coil domain located further downstream of the TRP signature domain (Fig. 1A). However, unlike some of the TRPM family members including TRPM2 (5, 6), TRPM7 (12, 13, 14), and TRPM6,2 hTRPM3 does not contain an enzyme domain in the C-terminal cytoplasmic region.
hTRPM3 is most similar to hTRPM1 with 57% identity and 67% similarity (Fig. 1, A and C). Greater homology (over 80% identity) was observed at the N terminus (between amino acids 1 and 1219). There is a 58 amino acid gap in the hTRPM3 sequences as shown in the alignment of hTRPM3 with hTRPM1. GENEWISEDB was used to look for possible exons at the corresponding genomic DNA region, and none was found. There are good splice junctions around the sequence gap, and the exon forced out by GENEWISEDB has no homology to TRPM1. Therefore, it is unlikely that any coding sequence was missed within that region.
hTRPM3 Gene Is Located at 9q-21.12An analysis of the genomic sequence of hTRPM3 (Fig. 1B) showed that the coding region spans 311 kb and is comprised of 24 exons. hTRPM3 gene is located between the two genomic markers, D9S1874 and D9S1807, and its chromosomal localization is 9q-21.12.
We identified five more splice variants from a human kidney cDNA library using the primers designed from the predicted coding sequence of hTRPM3 gene (Fig. 1B). A comparison with the genomic DNA sequence shows that the exon boundaries of all of the splice variants obey the gt-ag rule of the splice donor-donor-acceptor sites. We designate the splice variants as "TRPM af" according to their relative abundance, subject to the ratios of products formed from the PCR amplification. The following experiments were all performed using the "a" form.
hTRPM3 Is Expressed Selectively in Human KidneyFig. 2A illustrates the relative expression level of hTRPM3 among various human mRNA tissue sources by Northern analysis using a 645-bp hTRPM3-specific probe (Fig. 1A, corresponding to the region between two arrowheads). The transcripts of 8 kb corresponding to hTRPM3 are expressed predominately in kidney tissue. The hTRPM3 polypeptide was also expressed at lesser levels in the brain and testis. Consistent with the identification of several other splice variants, multiple species of hTRPM3 transcripts were also detected in the Northern blot.
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A similar expression pattern was observed by an independent method, quantitative RT-PCR. As shown in Fig. 2B, transcripts corresponding to hTRPM3 expressed predominately in kidney tissue and, at lesser levels, in brain, testis, and spinal cord. On an extended panel of human tissue mRNAs, it was demonstrated that within brain subregions, the highest levels of expression were found in the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus, and the substantia nigra (data not shown).
hTRPM3 mRNA expression in human kidney was further analyzed by in situ hybridization. hTRPM3 was localized to the cytoplasm of collecting tubular epithelium in the medulla, medullary rays, and periglomerular regions (Fig. 2C, i and v). Tubules in the medulla exhibited the most intense expression. Other tubular epithelia, e.g. proximal convoluted tubular epithelium, exhibited minimal expression. Expression patterns were compared with hTRMP3 sense mRNA-labeled human kidney sections as negative controls (Fig. 2C, iii and vi) and to human lysozyme antisense mRNA-labeled human kidney sections as positive controls (data not shown).
An analysis of hTRPM3 expression has also been made in mRNA isolated from various tumors and control tissues. Renal tumors showed a significant decrease (average of 80% lower) in hTRPM3 steady-state mRNA levels in the tumors compared with their matched normal kidney controls. Similarly, in testicular cancers, lower steady-state mRNA levels also were observed (data not shown). These data suggest that a loss of hTRPM3 expression might play a role in tumorigenesis.
Overexpressed hTRPM3 Can Be Detected at the Plasma Membrane in HEK 293 CellsThe complete open reading frame of hTRPM3 with a C-terminal HA tag was transiently transfected into HEK 293 cells to analyze the biological function. The expression of full-length protein was assessed with the immunoblot using an anti-HA antibody and detected as the expected size of 170 kDa (data not shown).
The cellular localization of HA-tagged hTRPM3 was detected using a fluorescein-conjugated anti-HA antibody and a laser-scanning confocal microscope. Anti-HA staining was found to be associated with the membrane marker CM-DiI, indicating hTRPM3 protein in or near the plasmalemmal compartment of transfected cells (Fig. 2D). Plasmalemmal localization is consistent with the function of the TRP family as Ca2+-permeable membrane protein. hTRPM3 was also observed in intracellular compartments, possibly resulting from overexpression in this heterologous expression system as observed with other ion channels (25).
hTRPM3 Mediates Ca2+ EntryTo assess the functional role of hTRPM3, we tested for Ca2+ permeability, a property common to most TRP channels. Cells transiently transfected with vector or hTRPM3 were loaded with the cytoplasmic Ca2+ indicator, Fluo-4. Intracellular Ca2+ was monitored using a FLIPR that measures real-time intracellular fluorescence changes. Initially, cells were maintained in a 1 mM Ca2+ solution, which is in the normal range of physiological conditions. After measuring base-line intracellular Ca2+ upon FLIPR addition of 1, 3, or 10 mM CaCl2 to the media resulted in a concentration-dependent increase in intracellular Ca2+ in hTRPM3-expressing cells (Fig. 3A, right panel). In contrast, vector-transfected cells showed minimal Ca2+ entry under the same experimental conditions (Fig. 3A, left panel). Non-transfected cells were indistinguishable from vector-transfected cells (data not shown). These results indicate that hTRPM3 is capable of mediating Ca2+ entry.
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To further address the mechanism of hTRPM3-mediated Ca2+ entry, Ca2+ addition experiments were performed on transfected cells incubated (30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (26). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added subsequently to the buffer, indicating that Ca2+ entry was mediated through endogenous SOCs in HEK 293 cells (Fig. 3B, left panel). In hTRPM3-transfected cells, the Ca2+ transients triggered by a similar Ca2+ treatment were much larger (Fig. 3B, right panel). In addition, Ca2+ entry observed in hTRPM3-transfected cells incubated in Ca2+-free media was greater than in 1 mM Ca2+ media (compared with Fig. 3A, right panel), indicating that hTRPM3-mediated Ca2+ entry was potentiated by store depletion.
The store-operated mechanism of hTRPM3-mediated Ca2+ entry was tested further by passively depleting Ca2+ stores with TG, an inhibitor of microsomal Ca2+-ATPase whose normal function is to pump ions from the cytosol back into the stores. The addition of 2 µM TG equivalently depleted Ca2+ stores in hTRPM3- and vector-transfected cells (Fig. 3C). Following store depletion with TG, the addition of Ca2+ to the buffer induced a much larger Ca2+ entry in hTRPM3 cells compared with the vector control cells (Fig. 3C, 14% increase measured at peak selected from t = 600660 s; hTRPM3, 19550 ± 226; vector, 17213 ± 413, n = 12).
Receptor-mediated Ca2+ entry was also more pronounced in hTRPM3-transfected cells. Carbachol (CCh) can activate an endogenous muscarinic receptor and trigger inositol 1,4,5-trisphosphate production, leading to the activation of SOCs in HEK 293 cells (27). The addition of 50 µM CCh caused a transient and rapid intracellular Ca2+ increase in both hTRPM3- and vector-transfected cells (Fig. 3D). After the receptor activation with CCh, the addition of Ca2+ to the buffer induced a much larger influx of Ca2+ into hTRPM3 cells as compared with vector control cells (Fig. 3D, 40% increase measured at peak selected from t = 660720 s; hTRPM3, 11067 ± 218; vector, 7879 ± 248; n = 12). These results show that after store depletion with TG or receptor activation with CCh, hTRPM3-transfected cells exhibit an increased Ca2+ influx when compared with control cells.
hTRPM3-mediated Ca2+ Entry Can Be Partially Blocked by Gd3+The Gd3+ is a nonselective Ca2+-permeable channel blocker that inhibits most known TRP channels. The effects of 100 µM Gd3+ on Ca2+ permeability were tested in vector- and hTRPM3-transfected cells. The minimal Ca2+ influx observed upon the addition of 10 mM Ca2+ to the cells (cells were incubated in the presence of 1 mM Ca2+) in vector-transfected cells (Fig. 4A) was strongly inhibited by 100 µM Gd3+. In contrast, 100 µM Gd3+ inhibited Ca2+ entry induced by adding 10 mM Ca2+ by 53% in hTRPM3-transfected cells (Fig. 4B). Gd3+ reduced fluorescence units in vector-transfected cells from 1470 ± 140 to -58 ± 8 and in hTRPM3-transfected cells from 6000 ± 322 to 2080 ± 199 (n = 12). Fluorescence values were measured 150 s after adding 10 mM Ca2+, and percent blockade was calculated as 1 - (FhTRPM3 - FVector in the presence of Gd3+)/(FhTRPM3 - FVector without blocker). The effects of Gd3+ on hTRPM3-mediated Ca2+ entry induced by 10 mM Ca2+ in the presence of TG or CCh were also examined. Cells were incubated in nominally Ca2+-free medium for TG and CCh experiments. Gd3+ inhibited Ca2+ entry by 51% after depletion of intracellular stores with TG (Fig. 4B). Gd3+ reduced peak fluorescence after 10 mM Ca2+ addition in vector-transfected cells from 26,444 ± 2410 to 1316 ± 60 and in hTRPM3-transfected cells from 37,676 ± 2425 to 6783 ± 250 (Fig. 4, C and D, respectively, n = 12). Gd3+ inhibited Ca2+ entry by 72% after depletion of intracellular stores with CCh. Gd3+ reduced peak Ca2+ fluorescence in vector-transfected cells from 9327 ± 466 to 453 ± 15 and in hTRPM3-transfected cells from 14,747 ± 988 to 1975 ± 79 (Fig. 4, E and F, respectively, n = 12). These results show that under identical conditions, the endogenous Ca2+ entry pathway was strongly blocked by the application of 100 µM Gd3+, whereas the hTRPM3- mediated pathway was partially blocked (53%) (Fig. 4, A and B). Stimulation of Ca2+ entry in hTRPM3-transfected cells in the presence of TG or CCh was also partially blocked by 100 µM Gd3+. These results are consistent with the hypothesis that hTRPM3 mediates a Ca2+ entry pathway that apparently is distinct from the endogenous Ca2+ entry pathways present in HEK 293 cells.
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DISCUSSION |
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hTRPM3 is the first member of the TRPM subfamily that has been shown to be modulated by store depletion. It is of interest to note that some members of the TRPC and TRPV also are modulated by store depletion (for review see Ref. 1). The known TRPM subfamily members exhibit diverse channel characteristics. TRPM1 and TRPM4a are Ca2+-permeable channels, but it is unclear whether they can be stimulated by store depletion (9). Distinct from TRPM4a, TRPM4b is activated directly by changes in intracellular Ca2+ without significant permeation of Ca2+ (10). TRPM2 is activated by ADP-ribose, NAD, and changes in redox status (5, 6, 7). TRPM7 is regulated by Mg2+-ATP and/or inositol 1,4,5-disphosphate (12, 13, 14). TRPM8 is activated by cold temperatures and cooling agents (16, 17). Therefore, in conjunction with a restricted tissue expression not observed with any other family members, hTRPM3 may have a unique biological function in humans.
We have shown that hTRPM3-mediated Ca2+ entry can be stimulated by lowering extracellular Ca2+ concentration (Fig. 3B), by passively depleting Ca2+ stores with TG treatment (Fig. 3C), and by treatment with CCh that activates G protein-coupled receptors (Fig. 3D). The store depletion-induced Ca2+ entry was significantly stimulated by adding 1 mM Ca2+ to the media in hTRPM3-expressing cells (Fig. 3B, right panel), whereas the addition of 1 mM Ca2+ to the media triggered only minimal hTRPM3 activity in cells of which intracellular Ca2+ stores were not depleted (compared with Fig. 3A, right panel). These results show that Ca2+ entry in hTRPM3-transfected cells was stimulated by store depletion, unlike some TRPC subfamily members such as TRPC3 and TRPC6, which show receptor-mediated activation independent of the store depletion (for review see Ref. 1).
The hTRPM3 gene maps to chromosome 9q-21.12 between the two markers, D9S1874 and D9S1807. Diseases that have been linked to this region include amyotrophic lateral sclerosis with frontotemporal dementia, early-onset pulverulent cataract, familial hemophagocytic lymphohistiocytosis, infantile nephronophthisis, and hypomagnesemia with secondary hypocalcemia (HSH). Given its selective expression in kidney, hTRPM3 could be considered a candidate gene for HSH, because the phenotype is a renal insufficiency. The chromosomal location of hTRPM3 is 600 kb downstream of an X;9 translocation breakpoint interval described for one patient with HSH (28). Recently, two groups (29, 30) reported that a new member of the TRPM subfamily, TRPM6, expressed in both intestinal tissues and kidney, is associated with HSH. Indeed, human TRPM3 is
4 million bp 5' to TRPM6 on 9q-21. Although TRPM6 has been implicated directly in HSH, this may not preclude the involvement of hTRPM3.
The kidney plays a major role in Ca2+ homeostasis. hTRPM3 could be involved in Ca2+ absorption directly because of its Ca2+ permeability. Indeed, the in situ hybridization analysis demonstrated that hTRPM3 is predominantly present in the collecting tubule, which has frequently been implicated in active transcellular Ca2+ reabsorption (for review see Ref. 31). Alternatively, hTRPM3 may function as a SOC that regulates Ca2+ absorption. In the kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone, and parathyroid hormone-related peptide through their respective G protein-coupled receptors and downstream SOCs (for review see Ref. 32). Future experiments using antisense RNA or knock-out mice may help define the functional role of hTRPM3 in the kidney.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 21-2.07, 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534. Tel.: 609-818-4293; Fax: 609-818-3239; E-mail: ning.lee{at}bms.com.
1 The abbreviations used are: SOC, store-operated Ca2+ channel; TRP, transient receptor potential; HEK, human embryonic kidney; TG, thapsigargin; HMM, hidden Markov model; FLIPR, fluorometric imaging plate reader; CCh, carbachol; hemagglutinin; RT, reverse transcriptase; Gd3+, lanthanide gadolinium; HSH, hypomagnesemia with secondary hypocalcemia.
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ACKNOWLEDGMENTS |
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REFERENCES |
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