Expression and Characterization of Human Transient Receptor Potential Melastatin 3 (hTRPM3)
2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês
10.1074/jbc.m211232200
ISSN1083-351X
AutoresNing Lee, Jian Chen, Lucy Sun, Shujian Wu, Kevin R. Gray, Adam Rich, Minxue Huang, Jun-Hsiang Lin, John N. Feder, Evan B. Janovitz, Paul Lévesque, Michael A. Blanar,
Tópico(s)Ion channel regulation and function
ResumoTransient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. The hTRPM3 gene is comprised of 24 exons and maps to human chromosome 9q-21.12. hTRPM3 is composed of 1555 amino acids and possesses the characteristic six-transmembrane domain of the TRP family. hTRPM3 is expressed primarily in kidney and, at lesser levels, in brain, testis, and spinal cord as demonstrated by quantitative RT-PCR and Northern blotting. In situ hybridization in human kidney demonstrated that hTRPM3 mRNA expression is predominantly found in the collecting tubular epithelium. Heterologous expression of hTRPM3 in human embryonic kidney cells (HEK 293) showed that hTRPM3 is localized to the cell membrane. hTRPM3-expressing cells exhibited Ca2+ concentration-dependent Ca2+ entry. Depletion of intracellular Ca2+ stores by lowering extracellular Ca2+ concentration and treatment with the Ca2+-ATPase inhibitor thapsigargin or the muscarinic receptor agonist carbachol further augmented hTRPM3-mediated Ca2+ entry. The nonselective Ca2+ channel blocker, lanthanide gadolinium (Gd3+), partially inhibited hTRPM3-mediated Ca2+ entry. 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. Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. The hTRPM3 gene is comprised of 24 exons and maps to human chromosome 9q-21.12. hTRPM3 is composed of 1555 amino acids and possesses the characteristic six-transmembrane domain of the TRP family. hTRPM3 is expressed primarily in kidney and, at lesser levels, in brain, testis, and spinal cord as demonstrated by quantitative RT-PCR and Northern blotting. In situ hybridization in human kidney demonstrated that hTRPM3 mRNA expression is predominantly found in the collecting tubular epithelium. Heterologous expression of hTRPM3 in human embryonic kidney cells (HEK 293) showed that hTRPM3 is localized to the cell membrane. hTRPM3-expressing cells exhibited Ca2+ concentration-dependent Ca2+ entry. Depletion of intracellular Ca2+ stores by lowering extracellular Ca2+ concentration and treatment with the Ca2+-ATPase inhibitor thapsigargin or the muscarinic receptor agonist carbachol further augmented hTRPM3-mediated Ca2+ entry. The nonselective Ca2+ channel blocker, lanthanide gadolinium (Gd3+), partially inhibited hTRPM3-mediated Ca2+ entry. 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. Intracellular Ca2+ plays a pivotal role in various cellular functions such as protein secretion, cell differentiation, cell division, and apoptosis. Ca2+ entry into cells can be mediated via voltage-dependent Ca2+ channels, ligand-gated Ca2+ channels, nonselective cation channels, and the relatively less characterized class store-operated Ca2+ channels (SOC). 1The 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. SOC mediate Ca2+ entry from the extracellular space following Ca2+ release from the intracellular stores to generate sustained increases in intracellular Ca2+ concentration and replenish the internal Ca2+ stores. The molecular mechanism of SOC activation and the molecular identity of SOC remain elusive. Transient receptor potential (TRP) channels, an emerging class of the Ca2+-permeable cation channel superfamily, are probable candidates for SOC (for review see Ref. 1Harteneck C. Plant T D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Following the identification of the founding member of this family, dTRP, which is from a Drosophila mutant with abnormal visual signal transduction (2Hardie R.C. Minke B. Neuron. 1992; 8: 643-651Abstract Full Text PDF PubMed Scopus (595) Google Scholar), 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) (3Montell C Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). 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 (4Duncan L.M. Deeds J. Hunter J. Shao J. Holmgren L.M. Woolf E.A. Tepper R.I. Shyjan A.W. Cancer Res. 1998; 58: 1515-1520PubMed Google Scholar). TRPM2 is a Ca2+-permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (5Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (772) Google Scholar, 6Sano Y. Inamura K. Miyake A. Mochizuki S. Yokoi H. Matsushime H. Furuichi K. Science. 2001; 293: 1327-1330Crossref PubMed Scopus (393) Google Scholar), and changes in redox status (7Hara Y. Wakamori M. Ishii M. Maeno E. Nishida M. Yoshida T. Yamada H. Shimizu S. Mori E. Kudoh J. Shimizu N. Kurose H. Okada Y. Imoto K. Mori Y. Mol. Cell. 2002; 9: 163-173Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar). The TRPM2 gene is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holosencephaly (8Nagamine K. Kudoh J. Minoshima S. Kawasaki K. Asakawa S. Ito F. Shimizu N. Genomics. 1998; 54: 124-131Crossref PubMed Scopus (216) Google Scholar). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca2+-permeable channel (9Xu X.-Z.S. Moebius F. Gill D.L. Montell C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10692-10697Crossref PubMed Scopus (191) Google Scholar); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca2+ (10Launay P. Fleig A. Perraud A.-L. Scharenberg A.M. Penner R. Kinet J.-P. Cell. 2002; 109: 397-407Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (11Prawitt D. Enklaar T. Klemm G. Gartner B. Spangenberg C. Winterpacht A. Higgins M. Pelletier J. Zabel B. Hum. Mol. Genet. 2000; 9: 203-216Crossref PubMed Scopus (114) Google Scholar). 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 (12Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (648) Google Scholar, 13Nadler 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 (825) Google Scholar, 14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (466) Google Scholar). TRPM8 is up-regulated in prostate cancer and other malignancies (15Tsavaler L. Shapero M.H. Morkowski S. Laus R. Cancer Res. 2001; 61: 3760-3769PubMed Google Scholar). Recently, it has been shown to be a receptor that senses cold stimuli (16McKemy D.D. Neuhausser W.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (2066) Google Scholar, 17Peier A.M. Moqrich A. Hergarden A.C. Reeve A.J. Andersson D.A. Story G.M. Earley T.J. Dragoni I. McIntyre P. Bevan S. Patapoutian A. Cell. 2002; 108: 705-715Abstract Full Text Full Text PDF PubMed Scopus (1810) Google Scholar). 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 (3Montell C Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). 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. Bioinformatics Analysis—The sequence homology search program BLAST and the gene-finding program Genewise/Wise2 package (18Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61086) Google Scholar, 19Bateman A. Berney E. Durbin R. Eddy S.R. Howe K.L. Sonnhammer E.L. Nucleic Acids Res. 2000; 28: 263-266Crossref PubMed Scopus (1237) Google Scholar) were used to identify novel ion channel sequences in the TRP family. Individual known ion channel protein sequences in the TRP family were used as probes in a TBLASTN analysis versus the human genome sequence. A multiple sequence alignment of known TRP family members was also generated using the ClustalW algorithm alignment program in the software, Vector NTI 5.5. A hidden Markov model (HMM) specific for the TRP family was then constructed using the HMMER-BUILD program in HMMER2.2 package from the above multiple sequence alignment (20Eddy S.R. Bioinformatics. 1998; 14: 755-763Crossref PubMed Scopus (4191) Google Scholar). This HMM model was then used to search the human genomic sequence data base using the GENEWISEDB program in Genewise. Results from the TBLASTN and GENEWISEDB searches were pooled. The high scoring hit segments from the data base search were extracted and searched back against nonredundant protein and patent sequence databases to determine novelty. For potential novel protein-encoding segments, the most similar protein sequence hits were used as templates to predict putative exons from the genomic sequence using the GENEWISEDB program. From this analysis, exons encoding potential novel ion channels were identified. To extend the 5′ and 3′ sequences of putative novel ion channel molecules, the genomic regions surrounding the matching exons from genomic contigs were analyzed using GENSCAN and FGENESH programs to generate de novo exons (21Burge C. Karlin S. J. Mol. Biol. 1997; 268: 78-94Crossref PubMed Scopus (3166) Google Scholar, 22Salamov A.A. Solovyev V.V. Genome Res. 2000; 10: 516-522Crossref PubMed Scopus (849) Google Scholar). Based on these analyses, one of the predicted full-length protein coding sequences of novel human ion channel related genes was cloned for further study. 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 GenBank™ accession numbers are as follows: hTRPM1 (NM_002420), hTRPM2 (NM_003307), hTRPM4 (NM_017636), hTRPM5 (NM_014555), hTRPM6, 2N. Lee, unpublished data. hTRPM7 (XM_030709), and hTRPM8 (NM_024080). Cloning of hTRPM3—Using the predicted exon genomic sequence from BAC AL358786, 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-PCR—A 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 Analysis—Human 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 × 106 cpm/ml 32P-labeled probe. Autoradiography was performed for 1 week at -70 °C. In Situ Hybridization—Human 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) (GenBank™ accession number AA588081). 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 (23Dambach D.M. Watson L.M. Gray K.R. Durham S.K. Laskin D.L. Hepatology. 2002; 35: 1093-1103Crossref PubMed Scopus (232) Google Scholar). 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 × 106 cpm/slide) for 16–20 h at 60 °C. Following hybridization, slides were rinsed in 4× SSC/50% formamide and 4× SSC, treated with RNase A (20 μg/ml, Invitrogen) at 37 °C, washed through increasing stringent solutions to a final high stringency wash in 0.1× 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 hTRPM3—HEK 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 ×63 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 (FLIPR™, 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 510–570-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. hTRPM3 Is a Novel Member of the TRP Channel Family— Human BAC AL358786 was found to cover a partial open reading frame of a novel TRP family gene based on the BLASTTN and GENEWISEDB searches. TRPM1, the most similar protein for this novel TRP gene, was used as a template to predict additional exons from the same genomic sequence using the GENEWISEDB program. To extend the 5′ and 3′ sequences of the TRP gene, the genomic regions surrounding the matching exons in genomic sequence NT_008306 were analyzed using GENSCAN and FGENESH programs to generate de novo exons (21Burge C. Karlin S. J. Mol. Biol. 1997; 268: 78-94Crossref PubMed Scopus (3166) Google Scholar, 22Salamov A.A. Solovyev V.V. Genome Res. 2000; 10: 516-522Crossref PubMed Scopus (849) Google Scholar). A full-length TRP family gene was discovered using these analyses. This computational prediction was then confirmed experimentally. A cDNA was isolated from a human kidney library by using the putative exon sequences as PCR templates. We refer to this gene as hTRPM3 (3Montell C Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). The C-terminal sequence is nearly identical to a previously reported cDNA fragment KIAA1616 (24Nagase T. Kikuno R. Nakayama M. Hirosawa M. Ohara O. DNA Res. 2000; 7: 273-281PubMed Google Scholar) and later denoted as TRPM3 (3Montell C Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). As compared with KIAA1616, our sequence contains 566 additional amino acids at the N terminus of KIAA1616 (Fig. 1B). With the following experimental evidence, we believe that we have the full-length functional hTRPM3. 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 748–959); 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 (5Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (772) Google Scholar, 6Sano Y. Inamura K. Miyake A. Mochizuki S. Yokoi H. Matsushime H. Furuichi K. Science. 2001; 293: 1327-1330Crossref PubMed Scopus (393) Google Scholar), TRPM7 (12Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (648) Google Scholar, 13Nadler 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 (825) Google Scholar, 14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (466) Google Scholar), 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-21.12—An 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 a–f" 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 Kidney—Fig. 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. 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 Cells—The 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 (25Marshall J. Molloy R. Moss G.W. Howe J.R. Hughes T.E. Neuron. 1995; 14: 211-215Abstract Full Text PDF PubMed Scopus (220) Google Scholar). hTRPM3 Mediates Ca2+Entry—To 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. 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 (26Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalie A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Crossref PubMed Scopus (274) Google Scholar). 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-transf
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