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Expression of CaT-like, a Novel Calcium-selective Channel, Correlates with the Malignancy of Prostate Cancer

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m009895200

1083-351X

Ulrich Wissenbach, Barbara A. Niemeyer, Thomas Fixemer, Arne Schneidewind, Claudia Trost, Adolfo Cavalié, Katrin Reus, Eckart Meese, Helmut Bonkhoff, Veit Flockerzi,

Cardiac electrophysiology and arrhythmias

The regulation of intracellular Ca2+ plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies. The regulation of intracellular Ca2+ plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies. epithelial Ca2+ channel base pair polymerase chain reaction green fluorescent protein human embryonic kidney phosphate-buffered saline kilobase pair The link between ion channels and disease has received widespread attention in the last few years as mutations in several ion channels have been shown to be responsible for various forms of neurological disorders (1Weinreich F. Jentsch T.J. Curr. Opin. Neurobiol. 2000; 10: 409-415Crossref PubMed Scopus (20) Google Scholar, 2Ashcroft F.M. Ion Channels and Disease. Academic Press, London1999: 352Google Scholar). Whereas many of these mutations affect well characterized channels of the nervous system, little is known about the situation in non-excitable cells. One new superfamily of channels of widespread expression and function include channels of the Trp family. The prototypical members of this family of six transmembrane domain channel subunits come from the visual system of Drosophilawhere they have been shown to be responsible for the light-activated cationic conductance changes (3Scott K. Zuker C. Curr. Opin. Neurobiol. 1998; 8: 383-388Crossref PubMed Scopus (63) Google Scholar). Other members of these growing family of ion channels include osmo- and mechanosensitive ion channels (4Colbert H.A. Smith T.L. Bargmann C.I. J. Neurosci. 1997; 17: 8259-8269Crossref PubMed Google Scholar, 5Walker R.G. Willingham A.T. Zuker C.S. Science. 2000; 287: 2229-2234Crossref PubMed Scopus (546) Google Scholar), channels responsible for pain and heat perception like the vanilloid receptors (6Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7307) Google Scholar, 7Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1292) Google Scholar), and channels involved in agonist/receptor activated cation influx (8Putney J.W.J. McKay R.R. BioEssays. 1999; 21: 38-46Crossref PubMed Scopus (359) Google Scholar) into cells such as Trp-1 to Trp-7. Also new on the scene are the epithelial Ca2+ channel, ECaC1 (also ECaC1 (9Hoenderop J.G. van der Kemp A. Hartog A. van de Graaf S. van Os C. Willems P.H. Bindels R.J. J. Biol. Chem. 1999; 274: 8375-8378Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar)), and the Ca2+ transport protein CaT1 (also ECaC2, (10Peng J.B. Chen X.Z. Berger U.V. Vassilev P.M. Tsukaguchi H. Brown E.M. Hediger M.A. J. Biol. Chem. 1999; 274: 22739-22746Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar)), implicated to play a role in the reabsorption of Ca2+ by the kidney (ECaC) and intestinal epithelial cells (ECaC and CaT1). Two other identified members of this family of Trp-related proteins, p120 and melastatin, have not yet been demonstrated to function as ion channels. One of these genes, p120 (11Jaquemar D. Schenker T. Trueb B. J. Biol. Chem. 1999; 274: 7325-7333Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), when overexpressed, appears to interfere with normal cell growth, whereas the second, melastatin (12Duncan 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), is abundantly expressed in benign cutaneous nevi but appears to be down-regulated in primary melanomas and, especially, in metastatic lesions. Here we report the cloning of a new human gene product that is structurally related to the rat CaT1 cDNA and that we tentatively called Ca2+ transport protein-like (CaT-L). Unlike CaT1 and ECaC, CaT-L is not expressed in the small intestine (CaT1, ECaC (10Peng J.B. Chen X.Z. Berger U.V. Vassilev P.M. Tsukaguchi H. Brown E.M. Hediger M.A. J. Biol. Chem. 1999; 274: 22739-22746Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar,13Hoenderop 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)), in colon (CaT1 (10Peng J.B. Chen X.Z. Berger U.V. Vassilev P.M. Tsukaguchi H. Brown E.M. Hediger M.A. J. Biol. Chem. 1999; 274: 22739-22746Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar)) and in the kidney (ECaC (9Hoenderop J.G. van der Kemp A. Hartog A. van de Graaf S. van Os C. Willems P.H. Bindels R.J. J. Biol. Chem. 1999; 274: 8375-8378Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 13Hoenderop 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)). CaT-L is abundantly expressed in the placenta, pancreatic acinar cells, and salivary glands. So far, little is known of the Ca2+ entry pathways in these tissues. The Ca2+-permeation properties of the CaT-L channel, shown here, renders CaT-L as a good candidate for secretion coupling in these tissues. Most interesting, the CaT-L transcripts are undetectable in benign prostate tissue but are present at high levels in locally advanced prostate cancer, metastatic lesions, and recurrent androgen-insensitive prostatic adenocarcinoma. Hence, molecular classification of prostate cancer subclasses and class prediction by monitoring the level of human CaT-L gene expression is feasible. In addition, functional characterization of the new Ca2+ channel suggests a possible link between Ca2+ signaling and prostate cancer progression. Total RNA was isolated from human placenta as described (14Wissenbach U. Schroth G. Philipp S. Flockerzi V. FEBS Lett. 1998; 429: 61-66Crossref PubMed Scopus (67) Google Scholar), and poly(A)+ RNA was obtained using poly(A)+ RNA spin columns (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. To obtain an oligo-(dT)-primed cDNA library, placenta poly(A)+ RNA was reverse-transcribed using the cDNA choice system (Life Technologies, Inc.), and the resulting cDNA was subcloned in λ-Zap phages (Stratagene, La Jolla, CA). After screening the library with the human expressed sequence tag 1404042 (GenBankTM), several cDNA clones were identified, isolated, and sequenced. Additional cDNA clones were isolated from two specifically primed cDNA libraries from a second placenta using primers corresponding to amino acids676HLSLPM and 271GPLTSTL of the CaT-L sequence (Fig. 1 a) and the 345-bp NcoI/BamHI and 596-bp EcoRI/SstI cDNA fragments of CaT-L as probes. Thirteen independent cDNA clones were sequenced on both strands. In addition the complete coding region of the CaT-L protein was amplified by PCR, using human cDNA isolated from placenta as template, and eight independent cDNA clones were sequenced on both strands. The nucleotide sequences of CaT-La and CaT-Lb have been deposited in DDBJ/EMBL/GenBankTM under the accession numbers AJ243500 and AJ243501, respectively. For Northern blot analysis 5 μg of human poly(A)+ RNA from human placenta and from prostate (obtained from patients undergoing transurethral prostatectomy because of benign prostatic hyperplasia) were separated by electrophoresis on 0.8% agarose gels and thereafter transferred to Hybond N nylon membranes (Amersham Pharmacia Biotech) as described (14Wissenbach U. Schroth G. Philipp S. Flockerzi V. FEBS Lett. 1998; 429: 61-66Crossref PubMed Scopus (67) Google Scholar). The membranes were hybridized in the presence of 50% formamide at 42 °C overnight. Alternatively, a human multiple tissue RNA blot (CLONTECH) was hybridized under the same conditions. The probe was a 345-bp EcoRI/BamHI fragment spanning the protein coding region of amino acid residues 528–643 of the CaT-L protein (Fig. 1 a), labeled by random priming with [α,32P]dCTP. Filters were exposed to x-ray films for 4 days. To obtain the recombinant dicistronic expression plasmid pdiCaT-L carrying the entire protein-coding regions of CaT-Lb and the GFP (15Prasher D.C. Eckenrode V.K. Ward W.W. Prendergast F.G. Cormier M.J. Gene (Amst.). 1992; 111: 229-233Crossref PubMed Scopus (1790) Google Scholar), the 5′- and 3′-untranslated sequences of the CaT-Lb cDNA were removed, and the consensus sequence for initiation of translation in vertebrates (16Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (1035) Google Scholar) was introduced immediately 5′ of the translation initiation codon; and the resulting cDNA was subcloned into the pCAGGS vector (17Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4659) Google Scholar), downstream of the chicken β-actin promoter. The internal ribosomal entry site derived from encephalomyocarditis virus (18Kim D.G. Kang H.M. Jang S.K. Shin H.S. Mol. Cell. Biol. 1992; 12: 3636-3643Crossref PubMed Scopus (104) Google Scholar), followed by the GFP cDNA containing a Ser-65 → Thr mutation (19Heim R. Cubitt A.B. Tsien R.Y. Nature. 1995; 373: 663-664Crossref PubMed Scopus (1545) Google Scholar), was then cloned 3′ to the CaT-Lb cDNA. The internal ribosomal entry site sequence allows the simultaneous translation of CaT-Lb and GFP from one transcript. Thus, transfected cells can be detected unequivocally by the development of green fluorescence. Human embryonic kidney (HEK) 293 cells (ATCC CRL 1573) were transfected with pdiCaT-L using lipofectamine (Qiagen, Hilden, Germany) as described (20Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Crossref PubMed Scopus (259) Google Scholar). For measuring [Ca2+]i, HEK cells were cotransfected with the pcDNA3-CaT-Lb and pcDNA3-GFP (21Philipp S. Wissenbach U. Flockerzi V. Putney J.W.J. Calcium Signaling. CRC Press, Inc., Boca Raton, FL2000: 321-342Google Scholar) in a ratio of 4:1. To obtain pcDNA3-CaT-Lb the entire protein coding region of CaT-Lb including the consensus sequence for initiation of translation in vertebrates (16Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (1035) Google Scholar) was subcloned into the pcDNA3 vector (Invitrogen, Groningen, Netherlands). Measurements of [Ca2+]i and patch clamp experiments were carried out 2 days and 1 day after transfection, respectively. The chromosomal localization of the human CaT-L gene was performed using NIGMS somatic hybrid mapping panel 2 (Coriell Institute, Camden, NJ) described previously (22Drwinga H.L. Toji L.H. Kim C.H. Greene A.E. Mulivor R.A. Genomics. 1993; 16: 311-314Crossref PubMed Scopus (164) Google Scholar, 23Dubois B.L. Naylor S.L. Genomics. 1993; 16: 315-319Crossref PubMed Scopus (106) Google Scholar) and primers corresponding to amino acids115YEGQTA and 158NLIYFG of the CaT-L sequence (Fig. 3 a). Patch clamp recordings on single transfected cells were performed at 22–25 °C in the tight seal whole-cell configuration using fire-polished patch pipettes (3–10 MΩ uncompensated series resistance) 2 days after transfection. Pipette and cell capacitance were electronically canceled before each voltage ramp. Membrane currents were filtered at 1.5 kHz and digitized at a sampling rate of 5–10 kHz. To analyze transfected cells, currents were recorded with an EPC-9 patch clamp amplifier controlled by Pulse 8.3 software (HEKA Electronics). The pipette solution contained (in mm) the following: 140 aspartic acid, 10 EGTA, 10 NaCl, 1 MgCl2, 10 Hepes (pH 7.2 with CsOH). The bath solution contained (in mm) the following: 110 NaCl, 10 CsCl, 2 MgCl2, 50 mannitol, 10 glucose, 20 Hepes (pH 7, 4 with CsOH) and 2 CaCl2, or no added CaCl2(−Ca2+ solution). Divalent free bath solution contained (in mm) the following: 116 NaCl, 10 CsCl, 50 mannitol, 10 glucose, 20 Hepes, 1 EGTA (pH 7, 4 with CsOH) and bath solution without Na+ contained 110N-methyl-d-glucamine instead of NaCl. Whole-cell currents were recorded every second by applying 200-ms voltage clamp ramps from −100 to +100 mV from a holding potential of either −40 or +70 mV. The holding potential of +70 mV, which reduces Ca2+influx, in combination with high internal EGTA was used to minimize Ca2+ dependent feedback mechanisms. Data are given as mean ± S.E. Values were not corrected for liquid junction potentials. Measured currents were normalized to cell capacitance,i.e. −25.3 ± 0.4 pA/pF for CaT-L-transfected cells (n = 12) and −1.56 ± 0.54 pA/pF for GFP controls (n = 6) at −80-mV ramp potential in normal bath solution. Measurements of [Ca2+]i in single HEK cells were performed with a digital imaging system (T.I.L.L. Photonics). Cells grown on coverslips were loaded with 4 μm fura-2/AM (Molecular Probes, Eugene, OR) for 60 min at 37 °C in minimal essential medium containing 10% fetal calf serum. Cells were washed three times with 300 μl of buffer containing 115 mm NaCl, 2 mmMgCl2, 5 mm KCl, 10 mm Hepes (pH 7.4). Nominal Ca2+-free solutions contained ∼2 μm Ca2+. [Ca2+]i was calculated from the fluorescence ratios obtained at 340 and 380 nm excitation wavelengths as described (24Garcia D.E. Cavalié A. Lux H.D. J. Neurosci. 1994; 14: 545-553Crossref PubMed Google Scholar). Experiments were repeated three times. Sense and antisense oligodeoxynucleotides corresponding to the amino acid residues11LILCLWSK, 637QDLNRQRI, and651FHTRGSED of the CaT-L sequence (Fig. 1 a) were synthesized. Using the BLAST sequence similarity search tool provided by the National Center for Biotechnology Information (Bethesda, MD), the antisense sequences show maximal similarity of <71% to sequences in the GenBankTM data base. The oligodeoxynucleotides used for hybridization were biotinylated at the 3′ end. The non-radioactive in situ hybridization method was carried out as described (25Bonkhoff H. Fixemer T. Hunsicker I. Remberger K. Am. J. Pathol. 1999; 155: 641-647Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) using formalin-fixed slices of 6–8 μm thickness. Briefly, the slices were deparaffinized, rehydrated in graded alcohols, and incubated in the presence of PBS buffer including 10 μg/ml proteinase K (Roche Molecular Biochemicals) for 0.5 h. After prehybridization, the slices were hybridized at 37 °C using the biotinylated deoxyoligonucleotides (0.5 pmol/μl) in the presence of 33% formamide for 12 h. Thereafter, the slices were rinsed several times with 2× SSC and incubated at 25 °C for 0.5 h with avidin/biotinylated tyramide peroxidase complex (ABC, Dako). After several washes with PBS buffer, the slices were incubated in the presence of biotinylated tyramide and peroxide (0.15% w/v) for 10 min, rinsed with PBS buffer, and additionally incubated with ABC for 0.5 h. The slices were then washed with PBS buffer and incubated in the presence of DAB solution (diaminobenzidine (50 μg/ml), 50 mm Tris/EDTA buffer, pH 8.4, 0.15% H2O2 in N,N-dimethylformamide, Merck). The reaction was stopped after 4 min by incubating the slides in water. Biotinylated tyramide was obtained by incubating NHS-LC biotin (sulfosuccinimidyl-6-[biotinimid]-hexanoate, 2.5 mg/ml, Pierce) and tyramine-HCl (0.75 mg/ml, Sigma) in 25 mmborate buffer (pH 8.5) for 12 h. The tyramide solution was diluted 1000-fold (v/v) in PBS buffer before use. Normal human tissue included placenta (n = 2), prostate tissue (n = 2), colon (n = 2), stomach (n = 2), lung (n = 2), kidney (n = 2), endometrium (n = 2), salivary glands (n = 2), pancreas (n = 2), and parathyroid glands (n = 2). Transurethral resections with benign prostatic hyperplasia were obtained from three patients without clinical and pathological evidence of malignancy. Prostate cancer tissue from five radical prostatectomy specimens was submitted for study. The pathological stages and grades included pT3b (n = 2), pT3a (n = 2), pT2b (n = 1), and primary Gleason grades 5 (n = 2), 4 (n = 2), and 3 (n = 2). Four foci of high grade prostatic intraepithelial neoplasia were identified in the radical prostatectomy specimens. Lymph node metastases were obtained from five staging lymphadenectomies without subsequent prostatectomy. The material further contained palliative transurethral resection specimens from five patients with recurrent androgen-insensitive adenocarcinomas after orchiectomy. All specimens were available as formalin-fixed paraffin-embedded tissue sections. Sequences were analyzed using the Heidelberg Unix Sequence Analysis Resources of the biocomputing unit at the German Cancer Research Center, Heidelberg. The phylogenetic distances of proteins were calculated with the Clustal/Clustree program (26Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar, 27Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56652) Google Scholar), and the similarity of protein sequences in pairs was calculated with the ClustalW algorithm (28Needleman S.B. Wunsch C.D. J. Mol. Biol. 1970; 48: 443-453Crossref PubMed Scopus (8137) Google Scholar). Photographs were scanned and processed using Corel Photo-Paint/Corel Draw and Adobe PhotoShop. In search of proteins distantly related to the Trp family of ion channels, a human expressed sequence tag (EST 1404042) was identified in the GenBankTMdata base using BLAST programs (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (73420) Google Scholar). This EST was used as a probe to screen oligo(dT) and additional specifically primed human placenta cDNA libraries. Several positive cDNA clones were isolated, sequenced, and found to contain the complete sequence of the EST 1404042 clone as well as additional 5′-sequences. These clones cover an mRNA of about 2.9 kb with an open reading frame of 2175 bases (Fig.1 a) encoding a protein of 725 amino acid residues that we tentatively called human Ca2+transport protein-like (CaT-L). Downstream of the CaT-L coding sequence an additional open reading frame has been postulated (GenBankTM accession numberX83877) 2N. Tomilin and V., Boyko, unpublished results. to represent a zinc finger type DNA-binding protein. The functional significance of this putative gene product is not known. Hydropathy analysis reveals a hydrophobic core in the CaT-L protein with six peaks likely to represent membrane-spanning helices (S1 to S6) and a putative pore region between S5 and S6 (Fig. 1 b). The hydrophobic core is flanked by long presumptive cytoplasmic domains at the N and C termini (Fig. 1 c). A similar topology has been proposed for the light-activated ion channels in theDrosophila compound eye, Trp and TrpL, and related nematode and mammalian gene products (21Philipp S. Wissenbach U. Flockerzi V. Putney J.W.J. Calcium Signaling. CRC Press, Inc., Boca Raton, FL2000: 321-342Google Scholar, 30Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 4: 159-166Abstract Full Text Full Text PDF Scopus (434) Google Scholar). The N-terminal region of the CaT-L protein (Fig. 1 c) contains six amino acid sequence motives (amino acid residues 45–69, 79–102, 116–140, 162–186, 195–219, and 239–263) related to the consensus sequence of ankyrin-like repeats (31Lux S.E. John K.M. Bennett V. Nature. 1990; 344: 36-42Crossref PubMed Scopus (437) Google Scholar). As shown in Fig. 1, a and d, amino acid sequence comparison places human CaT-L in close relationship to the rat intestine Ca2+ transport protein (CaT1 (10Peng J.B. Chen X.Z. Berger U.V. Vassilev P.M. Tsukaguchi H. Brown E.M. Hediger M.A. J. Biol. Chem. 1999; 274: 22739-22746Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar)) and the human renal epithelial Ca2+ channel (ECaC (9Hoenderop J.G. van der Kemp A. Hartog A. van de Graaf S. van Os C. Willems P.H. Bindels R.J. J. Biol. Chem. 1999; 274: 8375-8378Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 32Müller D. Hoenderop J.G. Meij I.C. van den Heuvel L.P. Knoers N.V. den Hollander A.I. Eggert P. Garcia-Nieto V. Claverie-Martin F. Bindels R.J. Genomics. 2000; 67: 48-53Crossref PubMed Scopus (127) Google Scholar)), sharing 90 (rat CaT1) and 77% (human ECaC) overall amino acid sequence identity. More distantly related members of this gene family include non-selective cation channels such as the rat vanilloid receptors Vr1 and VRL that share common amino acid sequence motives (21Philipp S. Wissenbach U. Flockerzi V. Putney J.W.J. Calcium Signaling. CRC Press, Inc., Boca Raton, FL2000: 321-342Google Scholar), although overall sequence identity is low (Vr1, 28%; VRL, 27%). To investigate CaT-L expression, Northern analysis was performed using poly(A)+ RNA from different human tissues and a 345-bpEcoRI/BamHI fragment of CaT-L cDNA as a probe (Fig. 2 a). We found that CaT-L transcripts of 3.0 kb are expressed in placenta, pancreas, and prostate. The size of these transcripts corresponds to the size of the cloned CaT-L cDNA (2902 bp). In addition, a shorter transcript of 1.8 kb is detectable in poly(A)+ RNA isolated from human testis, which may result from alternative mRNA processing in this tissue. No CaT-L transcripts were detected in heart, lung, liver, skeletal muscle, spleen, ovary, and leukocytes. Interestingly, no CaT-L transcripts could be detected in small intestine, where both CaT1 and ECaC transcripts have been detected, nor in colon and brain (CaT1) or in kidney (ECaC) where these transcripts are predominantly expressed. The lack of CaT-L expression in human kidney and intestine suggests that CaT-L does not serve the physiological functions in these tissues that have been associated with the ECaC and CaT1 proteins and that include intestinal and renal Ca2+ absorption. Therefore, CaT-L is unlikely to represent the human ortholog of rat CaT1. A human cDNA sequence of 446 bp has been deposited to the GenBankTM data base (accession number AJ277909) that is identical to the corresponding sequence reported here. This sequence has been postulated to represent part of human CaT1, but no data are available that support this suggestion. Interestingly a 115-bp fragment, tentatively called CaT-Like2 (CaT-L2), was amplified from human genomic DNA and sequenced. It encodes an amino acid sequence (Fig. 1 a) that shares 92% sequence identity with human CaT-L, 95% with human ECaC, and 81% with rat CaT1 sequences and may represent a part of an additional ECaC/CaT1-related channel. To characterize further the cell-specific expression of CaT-L transcripts, in situ hybridization experiments were performed, using various human tissue sections (Fig. 2,b–i) obtained from placenta (taken from a 10-week-old abort), pancreas (removed from patients with pancreatic cancer), salivary gland, colon, and kidney. In the placenta (Fig. 2 b) CaT-L transcripts are expressed in trophoblasts and syncytiotrophoblasts. In the pancreas (Fig. 2 c) CaT-L transcripts are restricted to acinar cells and are not detectable in ductal epithelial cells and Langerhans islets. No CaT-L expression was found in regions of pancreatic carcinomas (data not shown). In salivary glands, CaT-L expression occurs in subsets of myoepithelial cells (Fig.2 d). Corresponding to the results obtained by Northern blot analysis, no CaT-L transcripts could be detected in tissue sections of human colon (Fig. 2 h) and human kidney (Fig. 2 i). In addition no transcripts could be detected in stomach, endometrium, lung, and parathyroid gland (data not shown). Comparison of the DNA sequences of the various CaT-L cDNA clones obtained from a human placenta revealed that the sequences could be grouped into two classes, which differ in five nucleotide substitutions (Fig.3 a). Three of the five substitutions resulted in changes of the encoded amino acid residues, whereas two nucleotide substitutions were silent (a1080g andg1878a). The resulting two protein sequences that differ in three amino acid residues (R157C, V378M, and T681M) were called CaT-La and CaT-Lb (Figs. 1 a and 3 a). This finding was reproduced by isolating CaT-L a and b cDNA clones from a second placenta. In addition, PCR amplification of the full-length CaT-L cDNA from a third placenta yielded only the b variant when eight amplified full-length CaT-L cDNAs were subcloned and sequenced. The nucleotide substitutions may reflect a coupled polymorphism; alternatively, the underlying mRNAs of the two cDNA classes may be products of different gene loci or may arise by RNA editing. To distinguish between these possibilities, we first designed a primer pair common to both CaT-L a and b isoforms that flanked the silent substitution a1080g and the substitution that leads to the amino acid exchange V378M. We then PCR-amplified a DNA fragment of 458 bp from genomic DNA isolated from human T-lymphocytes. Both classes of DNAs were amplified, and both amplification products contained a common intron sequence of 303 bp (Fig. 3 b). The a1080gsubstitution in the CaT-Lb DNA generates a new recognition site for the restriction enzyme Bsp1286I (Fig. 3 b). Accordingly, genomic DNA isolated from blood cells of 12 healthy male human individuals was used as template to amplify the 458-bp DNA fragment, and the amplified DNAs were then incubated in the presence ofBsp1286I. In 11 out of 12 individuals the expected DNA fragments of the CaT-Lb variant could be identified, whereas one individual contained both a and b variants (Fig. 3 c). In summary, these findings suggest that the two CaT-L variants may be due to a coupled polymorphism of one gene locus. By using a monochromosomal hybrid mapping panel, this locus was assigned to human chromosome 7 (data not shown). To characterize the electrophysiological properties of CaT-L, CaT-L and GFP were co-expressed in HEK cells using the dicistronic expression vector pdiCaT-L. Whereas only small background currents were observed under control conditions (GFP alone), large inwardly rectifying currents could be recorded in CaT-L-transfected HEK cells after establishing the whole-cell configuration (Fig.4, a–e), indicating that CaT-L forms constitutively active ion channels. Switching the holding potential from the initial −40 to +70 mV, currents increased dramatically in size (Fig. 4, a and e). This increase in current size with a change in holding potential was not observed for sodium currents (at zero extracellular divalent ions) and indicates that CaT-L may be partially inactivated by intracellular Ca2+. For the following experiments voltage ramps were applied from a holding potential of +70 mV. Although the initial characterization of CaT-L currents was reminiscent of currents mediated by ECaC (33Hoenderop J.G. van der, K. A. Hartog A. van, O. C. Willems P.H. Bindels R.J. Biochem. Biophys. Res. Commun. 1999; 261: 488-492Crossref PubMed Scopus (97) Google Scholar, 34Vennekens R. Hoenderop J.G. Prenen J. Stuiver M. Willems P.H. Droogmans G. Nilius B. Bindels R.J. J. Biol. Chem. 2000; 275: 3963-3969Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 35Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Bindels R.J. Droogmans G. J. Physiol. (Lond.). 2000; 527: 239-248Crossref Scopus (149) Google Scholar), the sequence differences led us to a more detailed investigation of CaT-L selectivity. CaT-L-specific currents were completely abolished following removal of external Ca2+(Fig. 4, a and c) but slightly increased when external Na+ was removed (summarized in Fig.4 e). The ion exchange experiments and the inwardly rectifying current-voltage relationship with the rather positive reversal potential (E rev) provide strong evidence that CaT-L forms Ca2+-selective ion channels (Fig.4, a, c, and e). The Ca2+selectivity, as defined by the E rev, becomes even more evident (Fig. 4 c, inset) if the background current is subtracted (background current defined as the remaining current in the absence of Ca2+). The slight but consistent increase of current size in the absence of Na+ (Fig. 4 e) is largely due to a local perfusion effect as perfusion of unaltered bath solution (puff in Fig. 4 a) revealed a similar increase in current size and could indicate an activation mechanism partially mediated by shape changes. A feature of non-voltage-operated Ca2+-selective ion channels is their ability to conduct Na+ only if all external divalent cations, namely