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J. Biol. Chem., Vol. 275, Issue 36, 28186-28194, September 8, 2000

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A Rat Kidney-specific Calcium Transporter in the Distal Nephron^*

Ji-Bin Peng^§, Xing-Zhen Chen^§¶, Urs V. Berger^§, Peter M. Vassilev, Edward M. Brown, and Matthias A. Hediger^§**

From the  Membrane Biology Program and ^§ Renal and  Endocrine-Hypertension Divisions, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, December 1, 1999, and in revised form, June 29, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CaT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D_28K and the sodium calcium exchanger 1, NCX1, by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D_28K were colocalized in the same cells. CaT2 mediated saturable calcium uptake with a Michaelis constant (K_m) of 0.66 mM when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca²⁺. Sr²⁺ and Ba²⁺ but not Mg²⁺ also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd²⁺ elicited inward current in CaT2-expressing oocytes with a K_m of 1.3 mM. Cd²⁺, however, also potently inhibited CaT2-mediated Ca²⁺ uptake with an IC₅₀ of 5.4 µM. Ca²⁺ evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd²⁺ also provides a potential explanation for Cd²⁺-induced hypercalciuria and resultant renal stone formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calcium is a major component of the mineral phase of bones and teeth and serves in the body as both first and second messengers (1, 2). The Ca²⁺ concentrations in the blood and extracellular fluids are tightly controlled through the regulated translocation of calcium ions via intestinal absorption (3), renal reabsorption (4), and the formation and breakdown of bone. While bone serves as a reservoir for calcium within the body, intestine and kidney play crucial roles in maintaining day to day calcium balance, which is regulated by the calciotropic hormones, 1,25-dihydroxyvitamin D₃ and parathyroid hormone (PTH)¹ (5). It is well known that calcium absorption in intestine and its reabsorption in kidney involve similar processes that include both passive paracellular and active transcellular pathways (6-9). The paracellular pathway utilizes diffusion of calcium ions down their electrochemical gradient through the tight junctions between epithelial cells (10), while the transcellular pathway involves calcium transport across the epithelial cells (9). The transcellular pathway is a multistep process (11) comprising calcium entry across the apical membrane (12), transport of calcium across the cell from apical to basolateral membrane (13), and, finally, extrusion of calcium through the basolateral membrane (14). The entry of calcium into the cell is thought to be a carrier-mediated process. Intracellular translocation of calcium, in turn, is probably facilitated by the vitamin D-regulated, calcium-binding proteins, calbindin-D_9K (in mammalian intestine, mouse, and rat kidney) and calbindin-D_28K (in mammalian kidney) (15-17). The eventual extrusion of calcium ions at the basolateral cell surface takes place against an electrochemical gradient and is an energy-requiring process that is mediated mainly by the calcium ATPase and sodium-calcium exchanger (14, 18, 19).

In kidney, most calcium is reabsorbed in the proximal tubule and cortical thick ascending limb by a paracellular pathway involving diffusion of calcium down its electrochemical gradient, while most reabsorption in the distal convoluted tubule and connecting segment occurs via the transcellular route (5, 8, 19, 20). Although the active, transcellular reabsorption of calcium in the distal nephron only accounts for 5-10% of total calcium reabsorption along the nephron, it is responsive to PTH and 1,25-dihydroxyvitamin D₃ and plays an important role in the fine tuning of whole body calcium homeostasis (5, 8, 19, 20). Furthermore, more than one pathway exists in this nephron segment as indicated by the studies of Friedman and colleagues (21-24). In addition, the toxic heavy metal, cadmium, which causes hypercalciuria and renal stone disease in exposed workers (25, 26), shares with calcium the transcellular pathway for renal tubular transport in cells derived from the distal convoluted tubule (24).

Studies of the mechanism(s) underlying transcellular calcium transport have been hindered by the lack of a molecular understanding of how calcium enters the apical membrane of the epithelial cell until recently, when two groups isolated putative calcium carriers from rabbit kidney (ECaC) (27) and rat intestine (CaT1) (28), respectively. CaT1 from rat and ECaC from rabbit share 75% amino acid identity and exhibit similar functional properties that are consistent with their participation in the apical uptake mechanism for calcium. Both proteins share about 30% amino acid identity with the capsaicin receptor VR1 (a ligand-gated, pH-sensitive, and heat-activated cation channel) and its homologues (29). A splice variant of VR1 from kidney was reported to be a stretch-inhibitable, nonselective cation channel (30). Interestingly, CaT1 was found in rat small and large intestine but not in kidney, whereas ECaC was detected both in rabbit kidney and intestine by Northern analysis (27, 28). In order to identify the molecular identity of the rat kidney calcium carrier(s), we performed the following studies, which demonstrate that intestinal calcium absorption and renal calcium reabsorption via the transcellular route probably occur via distinct calcium carriers in the rat. We further demonstrate that cadmium is permeable to this carrier and is a potent blocker of calcium transport mediated by CaT2.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Library Screening-- The superficial kidney cortex  ZAP II cDNA library originally constructed from the CD1 (Charles River) rat was a generous gift from Dr. Alan S. L. Yu (Brigham and Women's Hospital and Harvard Medical School). The library contains approximately 5 × 10⁵ independent recombinants. The full-length cDNA of CaT1 was labeled with [³²P]dCTP using Ready-To-Go^TM DNA labeling beads (Amersham Pharmacia Biotech) and purified with ProbeQuant^TM G-50 microcolumns (Amersham Pharmacia Biotech). Screening of the library was performed at low stringency, employing hybridization overnight with 5× SSC, 2× Denhardt's solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA at 54 °C, followed by washing with 1× SSC for 4 h at 54 °C. The positive clones were in vivo excised into pBluescript II (SK) as described by the manufacturer, and both the 5'- and 3'-ends of the clones were sequenced. The full-length CaT2 cDNA was sequenced bidirectionally in the W. M. Keck facility at Yale University.

Northern Analysis-- Total RNA was prepared from male rats using guanidinium isothiocyanate and cesium trifluoroacetate (Amersham Pharmacia Biotech). Poly(A⁺) RNA was purified by oligo(dT)-cellulose chromatography. Poly(A⁺) mRNAs (3 µg/lane) were electrophoresed in 1% formaldehyde agarose gels and blotted onto nylon membranes. The full-length CaT2 cDNA was excised using EcoRV and PstI, labeled with [³²P]dCTP with Ready-To-Go^TM DNA labeling beads (Amersham Pharmacia Biotech) and purified with ProbeQuant^TM G-50 microcolumns (Amersham Pharmacia Biotech). The membrane was hybridized at 68 °C with ExpressHyb hybridization solution (CLONTECH, Palo Alto, CA) and 100 µg/ml denatured salmon sperm DNA for 16 h and then washed at 68 °C with 0.1× SSC, 0.1% SDS for 2 h. The film was exposed at 70 °C for 3 days.

RT-PCR-- First strand cDNAs were synthesized using the SuperScript Choice System for cDNA Synthesis (Life Technologies, Inc.) with oligo(dT)_12-18 primer using 2 µg each of poly(A⁺) RNA from rat kidney, duodenum, and cecum. 2 µl of each of the products (20 µl in total) were used as templates for PCR, which was carried out in a total volume of 50 µl. The CaT1- and CaT2-specific primers that were used were as follows: CaT1 sense primer, 5'-TGATCATCCTGCTGGTGGAGATTC-3' (nucleotides 1187-1210 of the CaT1 coding region); CaT1 antisense primer, 5'-CGAGGCAGCTTCCGTTCTAGCAT-3' (nucleotides 1826-1804 of the CaT1 coding region); CaT2 sense primer, 5'-CAAGAAGAAAGAGGCTCGTCAGATTCTA-3' (nucleotides 876-906 of the CaT2 coding region); CaT2 antisense primer, 5'-GCAAAAGCAAAATAGGTTAGGTGGTAC-3' (nucleotides 1667-1641 of the CaT2 coding region). The reactions were carried out using a GeneAmp PCR System 2400 (Perkin-Elmer) for 35 cycles. Each cycle consisted of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 68 °C for 30 s. A 60-s denaturation at 94 °C and 7 min extension at 72 °C were carried out before and after the 35 cycles, respectively. Product (7.5 µl) was loaded into each lane of a 1% agarose gel for electrophoresis.

In Situ Hybridization-- Complementary RNA probes for CaT2, sodium calcium exchanger, NCX1 (18), and calbindin-D_28K (31) were synthesized from either linearized expression plasmids containing 4.2 kb of the CaT2 sequence, 452 bp of the NCX1 sequence (18), or an 830-bp, PCR-generated calbindin-D_28K fragment with the coding region flanked by promoter sites. The CaT2 and NCX1 probes were labeled with digoxigenin (DIG)-UTP, and the calbindin-D_28K probe was labeled with either DIG- or FITC-UTP. The CaT2 probe was alkali-hydrolyzed to an average length of 500 bp. In situ hybridization was performed on cryosections (10 µm) of fresh-frozen tissue as described (32). The hybridization buffer consisted of 50% formamide, 5× SSC, 2% blocking reagent (Roche Molecular Biochemicals), 0.02% SDS, and 0.1% N-laurylsarcosine. The probe concentrations were approximately 100 ng/ml. For the double hybridization experiments, the CaT2 probe and the FITC-labeled calbindin-D_28K probe were combined during hybridization. Sections were immersed in slide mailers in hybridization solution and hybridized at 68 °C for 18 h. Sections were subsequently washed three times in 2× SSC and then twice for 30 min each in 0.2× SSC at 68 °C. The hybridized, DIG-labeled probes were visualized in the case of single labeling with alkaline phosphatase-conjugated, anti-DIG Fab fragments (1:5,000; Roche Molecular Biochemicals) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Roche Molecular Biochemicals). For the double labeling, the sections were incubated, in sequence, in (a) a mixture of alkaline phosphatase-conjugated, anti-DIG Fab fragments and mouse anti-FITC antibodies (1:500; Roche Molecular Biochemicals), (b) biotinylated anti-mouse antibodies (1:500; Vector), and (c) streptavidin-horse radish peroxidase (1:500; NEN Life Science Products). Tyramide signal amplification was then performed using an experimental formulation of biotin-tyramide ("Turbo-tyramide," courtesy of Mark Bobrow, NEN Life Science Products), the DIG-labeled probe was developed in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution overnight, and finally the FITC-labeled probe was detected through incubation in streptavidin-CY3, exactly as described by Berger and Hediger (32). Sections were finally rinsed in 100 mM Tris, pH 9.5, 150 mM NaCl, and 25 mM EDTA and coverslipped with Vectashield (Vector Laboratories).

Expression of CaT2 in Xenopus Oocytes-- The entire CaT2 cDNA was excised from the pBluescript II (SK) vector using SmaI and SalI and was subcloned into a Xenopus oocyte expression vector, pNWP, using the BglII (blunt-ended with Klenow enzyme) and SalI sites for in vitro transcription. In vitro transcription was performed with the mMESSAGE mMACHINE^TM T7 Kit (Ambion, Austin, TX). Defolliculated Xenopus laevis oocytes were injected with either 50 nl of water or synthetic complementary RNA (cRNA). Oocytes were assayed 2-3 days after injection of RNA.

⁴⁵Ca²⁺ Uptake Assay-- Standard uptake solution contained the following components: 100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ (including ⁴⁵Ca²⁺ at a final concentration of 10 µCi/ml; NEN Life Science Products), and 10 mM Hepes, pH 7.5. Uptake was performed at room temperature for 30 min, and oocytes were washed six times with ice-cold uptake medium. To determine the K_m for Ca²⁺, total CaCl₂ (including ⁴⁵Ca²⁺) concentrations in the uptake solutions were adjusted in a range of 0.05-5 mM. In experiments investigating inhibition of uptake, CdCl₂ was added to the uptake solution at concentrations of 0.0001-1 mM. Unless stated otherwise, data are presented as means obtained from at least three experiments with 7-10 oocytes per group using the S.E. as the index of dispersion. Statistical significance was defined as having a p value of less than 0.05 as determined by Student's t test.

Two-microelectrode Voltage Clamp-- The two-microelectrode voltage clamp experiments were performed using a method described previously (33) with a commercial amplifier (Clampator One, model CA-1B; Dagan Co., Minneapolis, MN) and pCLAMP software (version 8; Axon Instruments, Inc., Foster City, CA). An oocyte was introduced into the chamber containing Ca²⁺-free solution and was incubated for about 3 min before being clamped at 50 mV and performing measurements. In experiments involving voltage ramps, whole-cell current and voltage were recorded by digitizing at 300 µs/sample. When recording currents at a holding potential, digitization at 0.2 s/sample was used. Voltage ramping consisted of preholding at 150 mV for 200 ms to eliminate capacitive currents and a subsequent linear increase from 150 to +50 mV, with a total duration of 1.4 s. Voltage jumping consisted of 150-ms voltage pulses of between 140 and +60 mV, in increments of +20 mV. Steady state currents were obtained as the average values in the interval from 135 to 145 ms after the initiation of the voltage pulses.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of CaT2-- Screening of approximately 6 × 10⁵ plaques of a rat superficial kidney cortex cDNA library resulted in isolation of two independent clones, designated 11-2-1 (4.2 kb) and 1-2-2 (3.0 kb). The full-length cDNA of clone 11-2-1 was sequenced, whereas only the 5' and 3' portions of 1-2-2 were sequenced. Clone 1-2-2 contained the same 5' sequence but was 20 bp longer than the corresponding region of clone 11-2-1; the 3' sequence of clone 1-2-2 was also identical to that of 11-2-1 until nucleotide 3009 of the latter, at which point clone 1-2-2 has an additional 11 adenosine nucleotides. Restriction analysis carried out with SstI, BstXI, XbaI, BamHI, SmaI, PstI, EcoRI, EcoRV, HindIII, SalI, XhoI, ApaI, and KpnI (not shown) indicated that the other regions of clone 1-2-2 are identical to those of clone 11-2-1. Based on its sequence and functional similarities to CaT1 (see below), the protein encoded by clone 11-2-1 is designated CaT2, for Ca²⁺ transport protein subtype 2.

Primary Structure of CaT2-- The 4182-bp 11-2-1 cDNA clone contains an open reading frame of 2169 bp (from nucleotide 117 to 2285) encoding a protein of 723 amino acid residues (Fig. 1). Hydropathy analysis suggests that CaT2 is an integral membrane protein containing six transmembrane domains with an additional short hydrophobic stretch between transmembrane domains 5 and 6. The N-terminal region of CaT2 contains three ankyrin repeats at amino acid positions 72-94, 110-142, and 156-188 in an ankyrin repeat region (amino acids 38-255). CaT2 also contains two N-linked glycosylation sites at residues 351 and 539, which are located in the first and last extracellular loops, a single cAMP- and cGMP-dependent protein kinase phosphorylation site at residue 702, as well as five protein kinase C phosphorylation sites in its N-terminal cytoplasmic region (at residues 54, 138, 291, 292, and 311) and three protein kinase C sites in its C-terminal cytoplasmic region (at residues 647, 657, and 691) (Fig. 1). In addition, there are six and seven casein kinase II phosphorylation sites, as well as two and four N-myristoylation sites in its N- and C-terminal cytoplasmic regions, respectively.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence and predicted domain structure of CaT2. A, alignment of amino acid sequences of rat CaT2, rabbit ECaC, and rat CaT1. Ankyrin repeat domains are shown in open boxes, labeled as ANK REP, transmembrane segments are underlined, and the potential pore region is double underlined. Putative protein kinase A and protein kinase C phosphorylation sites and N-linked glycosylation sites are boxed and indicated by the star, diamond, and club symbols, respectively. The labeled ankyrin repeats, transmembrane domains, and pore region are the ones that are present in rat CaT2 if there are differences among the three proteins. B, predicted membrane topology and domain structure of rat CaT2. Ankyrin repeats are shown in gray, and the N-glycosylation site and the putative protein kinase A (star) and protein kinase C phosphorylation sites (arrows) are marked. Outer and inner leaflets of plasma membrane are indicated.

CaT2 shows 84.2 and 73.4% amino acid identities to ECaC (27) and CaT1 (28), respectively (Fig. 1). It also shows significant homologies (20-30% identities) to a number of additional proteins, including vanilloid receptor-like protein 1 (29), a growth factor-regulated channel (34), vanilloid receptor 1 (35), the Caenorhabditis elegans olfactory channel, OSM-9 (36), a stretch-inhibitable nonselective channel (a splice variant of mouse vanilloid receptor 1), SIC (30), C. elegans proteins with GenBank^TM accession nos. AAC04431, CAA98449, and CAA96603 (37), and an ankyrin-like protein with transmembrane domains that is specifically lost after oncogenic transformation of human fibroblasts (38). In addition to the similarity of the CaT2 ankyrin repeat region to ankyrin and a number of other ankyrin repeat-containing proteins, a short portion of the transmembrane region of CaT2 (amino acid residues 445-587) is similar to rat TRP2 (39) (25%, 445-578), mouse TRP2 (40) (25%, 445-478), bovine TRP homologue (41) (25%, 466-587), mouse TRP4 (GenBank^TM accession nos. AAC05179 and AAC05178) (25%, 468-587), Bos taurus capacitative calcium entry channel 1 (42) (25%, 468-587), a rat TRP homologue (43) (25%, 468-587), human TRP4 (GenBank^TM accession no. AAD51736) (25%, 468-587), Oryctolagus cuniculus and mouse capacitative calcium entry channel 2 (42) (23%, 468-587), mouse TRP5 (GenBank^TM accession no. AAC13550) (23%, 468-587), human polycystin-2 homolog (GenBank^TM accession no. AAD51859) (29%, 482-585), polycystin-L (PKDL, PKD2L) (44-46) (29%, 482-585), and human polycystic kidney disease 2-related protein (47) (29%, 482-585).

Tissue Distribution of CaT2-- Northern analysis using a full-length cDNA of clone 11-2-1 as a probe revealed a strong band of ~3 kb in rat duodenum and two moderate bands of ~3.5 and ~6.6 kb in kidney at high stringency (Fig. 2A). No signal was detected in the other tissues tested, including brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis. Since CaT1 is very abundant in duodenum and the DNA sequences of CaT1 and CaT2 are highly homologous, the 3-kb band in duodenum may have been generated by cross-hybridization of the CaT2 probe to CaT1 mRNA. To investigate this possibility, specific CaT1 and CaT2 primers were employed to perform RT-PCR on RNA from rat kidney, duodenum, and cecum. As shown in Fig. 2B, use of CaT2-specific primers amplified a fragment of the expected size (792 bp) for a CaT2-derived product in kidney but not in duodenum or cecum. Use of CaT1-specific primers, in contrast, amplified a band of the expected size (640 bp) for a CaT1-derived product in duodenum and cecum but not in kidney. Thus, the 3-kb band detected by Northern analysis in rat duodenum results from cross-hybridization of the CaT2 probe with CaT1 mRNA.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Tissue distribution of CaT2. A, Northern analysis of tissue distribution of CaT2 mRNA. Each lane was loaded with 2 µg of poly(A)+ RNA from the indicated adult rat tissues. B, detection of CaT2 and CaT1 expression in rat kidney and intestine by RT-PCR. 7.5 µl of each reaction was loaded and subjected to agarose gel electrophoresis as described under "Experimental Procedures." The expected length of the CaT2-derived fragment is 791 bp, while that derived from CaT1 639 bp.

Co-localization of CaT2 with Calbindin-D_28K and Sodium-Calcium Exchanger NCX1-- Hybridization of frozen sections of rat kidney to the CaT2, calbindin-D_28K, and NCX1 probes resulted in identical labeling patterns (Fig. 3, upper panel). A small subpopulation of tubules located mostly in the outer third of the cortex was labeled with each probe. Double in situ hybridization with the CaT2 and the calbindin-D_28K probes revealed that these two mRNAs are expressed by the same cells in the distal nephron (Fig. 3, lower panel). Previous studies have also demonstrated that NCX1 and calbindin-D_28K are colocalized in the same cells in the connecting segments and cortical collecting ducts of rabbit kidney (48).

View larger version (92K): [in this window] [in a new window]   Fig. 3.   CaT2 mRNA distribution in rat kidney and colocalization with calbindin D28K as shown by nonisotopic, in situ hybridization. A-C demonstrate that the CaT2 mRNA (A) is localized primarily in the outer third of the cortex in a pattern similar to those seen for calbindin-D28K (B) and NCX1 (C). D and E show that, in a section hybridized with both CaT2 and calbindin-D28K probes, CaT2 (D) and calbindin-D28K (E) are expressed in the same population of tubules, i.e. by distal convoluted and connecting tubules. Magnification bars, upper row, 2 mm; lower row, 100 µm.

Ca²⁺ Uptake in Oocytes Expressing CaT2-- Injection of oocytes with CaT2 cRNA enhances Ca²⁺ uptake by about 20-fold compared with that in water-injected control oocytes (Fig. 4A). CaT2-mediated Ca²⁺ uptake was linear at least for 1 h in oocyte (data not shown). Na⁺ and Cl had little effect on CaT2-mediated Ca²⁺ uptake, whereas low pH inhibited and high pH stimulated CaT2-mediated calcium uptake (Fig. 4A). As assessed using a two-microelectrode voltage clamp, CaT2-mediated Ca²⁺ influx is electrogenic. The addition of 1 mM Ca²⁺ to oocytes held at 50 mV evoked a large, transient increase in inward current to several hundred nA, followed by a rapid reduction to a plateau value of 20-50 nA (Fig. 4B, upper panel). The peak current (curve 2) is due to the activation of endogenous Ca²⁺-activated chloride channels (49), presumably as a result of the increased level of cytosolic Ca²⁺ beneath the membrane due to uptake of calcium by CaT2. The Cl current also contributes to the plateau phase, since it contains outward current at positive potentials that was caused by the influx of Cl (curve 3) (Fig. 4B, lower panel).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Ca2+ uptake and Ca2+-evoked current in oocyte expressing CaT2. A, ion and pH dependences of CaT2-mediated Ca2+ uptake. Uptakes were performed with standard medium, sodium-free medium in which NaCl was substituted with choline chloride (ChoCl), or low Cl (4 mM) medium in which NaCl was substituted with sodium cyclamate (NaCyc). The pH dependence of CaT2-mediated Ca2+ uptake was evaluated in standard uptake solution with varying pH. The endogenous Ca2+ uptake in the control oocytes in standard solution is shown (Control), while data shown were obtained by subtracting the uptake by water-injected oocytes from that in CaT2-injected oocytes under the same experimental conditions. Data are from three independent experiments and are expressed as mean ± S.E. B, Ca2+-evoked currents in oocytes expressing CaT2. Currents were recorded before and after Ca2+ addition in an oocyte expressing rat CaT2. Upper panel, inward current elicited by application of 1 mM Ca2+ at a holding potential of 50 mV. Lower panel, I-V curves obtained at the times indicated in the left panel, using a voltage ramp protocol.

Among the transition metal ions and lanthanide ions tested at 0.1 mM, including Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Gd³⁺, and La³⁺, only Cd²⁺ evoked a current in CaT2-expressing oocytes, while the others did not evoke currents that differed from those observed in control oocytes (not shown). Among the alkaline earth metal ions, Sr²⁺ and Ba²⁺ evoked inward currents in EGTA-injected oocytes (see below) expressing CaT2, which represented 19 ± 3% (n = 5) and 9 ± 3% (n = 5), respectively, of that elicited by Ca²⁺ under the same conditions (5 mM). Mg²⁺, however, did not evoke any current.

Currents Evoked by Ca²⁺ and Cd²⁺ in CaT2-expressing Oocytes Preinjected with EGTA-- Ca²⁺ and Cd²⁺ are known to share the same entry pathways in cells derived from distal convoluted tubules (24). We assessed, therefore, CaT2-mediated transport of Cd²⁺ and Ca²⁺ using the voltage clamp technique. Chelating intracellular calcium by injection of EGTA into oocytes to a final concentration of approximately 2 mM resulted in a 3-5-fold increase in calcium uptake (data not shown) and abolished the initial transient increase in inward current (Fig. 5A, upper panel). In addition, the outward current was nearly abolished in EGTA-injected oocytes (Fig. 5A, lower panel). Under the same conditions, EGTA-injected control oocytes demonstrated no detectable currents. Like Ca²⁺, Cd²⁺ evoked an inward current, without any initial transient increase in current, in EGTA-injected, CaT2-expressing oocytes (Fig. 5B, upper panel), which was not observed in control oocytes (not shown). The current evoked by 1 mM Cd²⁺ was 46 ± 7% (n = 5) of that elicited by 1 mM Ca²⁺ in CaT2-expressing, EGTA-injected oocytes. Therefore, CaT2 probably mediates the observed Ca²⁺- or Cd²⁺-evoked current in EGTA-injected oocytes (Fig. 5, upper panels), and the resultant current-voltage (I-V) curves (Fig. 5, lower panels) may reflect the nature of Ca²⁺ or Cd²⁺ influx via CaT2. The I-V curves (Fig. 5, lower panels) also indicate that CaT2-mediated Ca²⁺ or Cd²⁺ influx was enhanced at hyperpolarized potentials.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Ca2+- or Cd2+-evoked currents in oocytes expressing CaT2 that were preinjected with EGTA. A, Ca2+-evoked currents recorded in an EGTA-injected oocyte expressing rat CaT2. Upper panel, inward current due to the addition of 1 mM Ca2+ at 50 mV. Lower panel, I-V curves obtained at the times indicated in the upper panel, using a voltage ramp protocol. B, Cd2+-evoked currents recorded in an EGTA-injected oocyte expressing rat CaT2. Upper panel, inward current evoked by the addition of 1 mM Cd2+ at 50 mV. Lower panel, I-V curves obtained at the times indicated in the left panel, using a voltage ramp protocol. Data shown in A and B are from the same oocyte injected with 50 nl of 50 mM EGTA at least 2 h prior to measurements

Cd²⁺ Effects on CaT2-mediated Ca²⁺ Transport-- Cd²⁺ is a potent blocker of CaT1-mediated Ca²⁺ uptake (28). Therefore, we tested the effect of Cd²⁺ on CaT2-mediated Ca²⁺ influx. CaT2-mediated Ca²⁺ uptake was saturable with a Michaelis constant (K_m) of 0.66 ± 0.15 mM in oocytes that were not injected with EGTA (Fig. 6A). In experiments performed under similar conditions, Cd²⁺-evoked currents were also saturable with a K_m of 1.3 ± 0.15 mM for Cd²⁺. Comparable results were also obtained using oocytes injected with EGTA. Interestingly, 10 µM Cd²⁺, which generated no detectable current by itself, produced a 70% inhibition of the current evoked by 1 mM Ca²⁺ in EGTA-injected, CaT2-expressing oocytes (Fig. 6C). The IC₅₀ for the inhibitory effect of Cd²⁺ on Ca²⁺ uptake was 5.4 ± 1.4 µM (Fig. 6D), which was consistent with the result obtained using voltage clamp (Fig. 6C). The substantial difference between the K_m for CaT2-mediated Cd²⁺ uptake and its IC₅₀ for inhibition of Ca²⁺ uptake suggests that there may be more than one binding site for Cd²⁺.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Inhibitory effect of Cd2+ on CaT2-mediated Ca2+ transport. A, concentration dependence of Ca2+ uptake mediated by rat CaT2. Uptakes were performed at varying Ca2+ concentrations (0.02- 5 mM) in standard uptake medium with cRNA- or water-injected oocytes. The duration of uptake was 30 min (within the time range when uptake is linear), and the oocytes were washed six times with ice-cold uptake solution. Data are from three independent experiments and are expressed as mean ± S.E. B, concentration dependence of Cd2+-evoked currents mediated by rat CaT2. Peak values of Cd2+-evoked currents were recorded at varying concentrations (0.10-10 mM) in standard solution without Ca2+. Data shown were obtained from a representative oocyte expressing CaT2 with a holding potential at 50 mV. C, effect of Cd2+ on Ca2+-evoked current in a CaT2-expressing oocyte. Shown are currents evoked by 1 mM Ca2+, 0.01 mM Cd2+, and a combination of both 1 mM Ca2+ and 0.01 mM Cd2+ in an oocyte expressing CaT2 that was preinjected with EGTA. D, effect of Cd2+ on Ca2+ uptake in CaT2-expressing oocytes. Uptake experiments were performed in standard Ca2+ solution with 1 mM Ca2+ containing 10 µCi/ml 45Ca2+ and varying concentrations of Cd2+ (0.1-1000 µM). Data are from three independent experiments and are expressed as mean ± S.E.

Effect of Voltage-dependent Calcium Channel Blockers on CaT2-mediated Ca²⁺ Transport-- Voltage-dependent calcium channels (VDCC) have been found in kidney cells (21-23). The PTH- stimulated pathway of Ca²⁺ uptake in cells from cortical thick ascending limbs and distal convoluted tubules is sensitive to dihydropyridine-type agonists and antagonists of the voltage-dependent calcium channels (22). A PTH-stimulated pathway of Cd²⁺ uptake in the distal convoluted tubule was also found to be dihydropyridine-sensitive (24). However, ECaC was reported to be insensitive to calcium channel blockers (27), and CaT1 was only slightly inhibited by verapamil and diltiazem (28). Under voltage clamp, Ca²⁺-evoked currents in CaT2-injected oocytes were slightly modulated by antagonists and an activator of VDCC. Namely, in the presence of the VDCC antagonists, nifedipine, (±)-verapamil, and diltiazem, and the VDCC agonist, (±)-Bay K 8644, each at 0.1 mM, the currents evoked by 1 mM Ca²⁺ were 88.2 ± 0.9 (n = 9), 96.0 ± 1.8 (n = 6), 91.7 ± 3.5 (n = 7) and 93.5 ± 1.7% (n = 7) of those observed in the absence of these agents (Fig. 7A). Similar results were also obtained in studies of Ca²⁺ uptake over a 30-min time period (not shown). The modest effects of these agents were enhanced by preincubation (Fig. 7B), suggesting that the binding of the VDCC antagonists/agonist to CaT2 is a relatively slow process.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Inhibitory effect of voltage-dependent calcium channel blockers or a VDCC activator on calcium uptake. A, effects of the VDCC activator, (±)-Bay K 8644, or the VDCC blockers, nifedipine (N), (±)-verapamil (V), or diltiazem (D), on Ca2+-evoked currents in a CaT2-expressing oocyte. All of the solutions contained 1% (v/v) of ethanol as a vehicle control. The Ca2+ was at 1 mM, and the compounds were tested at 0.1 mM. B, Ca2+-evoked currents in a CaT2-expressing oocyte showed a greater degree of inhibition following preincubation with (±)-verapamil (0.1 mM) for 2 min prior to the addition of Ca2+ (0.2 mM).

Feedback Inhibition of CaT2 as a Result of Cellular Uptake of Ca²⁺-- Increases in intracellular Ca²⁺ are known to attenuate the activities of VDCC, TRPs, PC3, and ECaC. Therefore, we examined the effect of Ca²⁺ uptake on transport of Ca²⁺ by CaT2. Fig. 8A shows that increasing concentrations of extracellular Ca²⁺ cause progressive inhibition of calcium uptake in CaT2-expressing oocytes, as demonstrated by the amplitude-time profile of the currents evoked by 0.125, 0.250, and 1 mM Ca²⁺. This inhibitory effect appears to be a relatively slow process, perhaps because it requires chelation of Ca²⁺ transported into the oocytes via CaT2 by EGTA close to the site of Ca²⁺ influx, followed by equilibration with the larger pool of EGTA throughout the cytoplasm of the oocyte. In contrast, the inhibitory action of Cd²⁺ on Ca²⁺ uptake occurred much more rapidly, presumably because Cd²⁺ acts directly on the CaT2 pore region (Fig. 8B).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Inhibitory effects of intracellular Ca2+, verapamil, and Cd2+ on CaT2-mediated Ca2+ transport. A, inhibitory effects of Ca2+ transported into the oocytes by CaT2 were observed upon prolonged application of Ca2+ at 0.25 and 1 mM but not at 0.125 mM in an oocyte expressing CaT2 preinjected with EGTA. Note that currents diminished after reaching their peak values more rapidly at the higher levels of extracellular calcium, presumably as a result of more Ca2+ ions being transported intracellularly by CaT2. B, a comparison of the inhibitory effects of (±)-verapamil, Cd2+, and elevated levels of extracellular Ca2+ on CaT2-mediated Ca2+ transport.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression cloning of calcium channels/transporters from rabbit kidney (ECaC) and rat intestine (CaT1) has provided a potential molecular basis for calcium entry into the epithelial cells of kidney and intestine (27, 28). It is not surprising that these two proteins are similar in their primary (75% amino acid identity) and predicted secondary structures, since the overall process of transcellular calcium transport is similar in intestine and kidney (8). However, a complete understanding of the structural and functional relationships of these two proteins to one another requires their characterization in a single species using identical experimental approaches, an approach that we have taken here.

By screening a rat kidney cortex library, we obtained two clones that appear to have identical coding regions. The deduced amino acid sequence of one of these, designated CaT2, shows a high degree of identity to those of both ECaC and CaT1 (84.2 and 73.4%, respectively). CaT2 could be the rat homologue of ECaC, since it shares a high degree of sequence identity with ECaC and exhibits properties similar to those of ECaC and CaT1 in terms of its saturable Ca²⁺ uptake kinetics, pH sensitivity, and permeability to alkali earth metals. However, CaT2 and ECaC differ in terms of their relative levels of expression in intestine and their affinities for Ca²⁺. Furthermore, CaT2 is both sensitive and permeant to Cd²⁺, a property has not yet been evaluated for ECaC.

Although bands were detected in both rat duodenum and kidney by high stringency Northern analysis using a CaT2-specific probe (Fig. 2A), the RT-PCR data (Fig. 2B), acquired using CaT1- and CaT2-specific primer pairs, indicate that CaT1 is intestine-specific and CaT2 is kidney-specific in the rat. The reason why CaT2 mRNA was not detectable in rat kidney when using a CaT1 probe (28) may simply reflect the much greater abundance of CaT1 mRNA in duodenum relative to that of CaT2 mRNA in the kidney.

ECaC, in contrast, was reported to be expressed in rabbit kidney, intestine, and placenta (27). Three reasons may explain the apparent differences in tissue distribution between CaT2 and ECaC: (a) CaT2 may not be the rat homologue of ECaC; (b) there may be species differences in tissue-specific expression of CaT isoforms; and (c) the signal detected in rabbit intestine with the ECaC probe may be due to the cross-hybridization with mRNA of the rabbit homologue of CaT1.

CaT2 mRNA was detected in rat kidney cortex with an expression pattern similar to that of calbindin-D_28K and the sodium-calcium exchanger, NCX1 (Fig. 3, upper panel). Co-expression of CaT2 mRNA with calbindin-D_28K in the same cells was further demonstrated by double labeling of CaT2 and calbindin-D_28K mRNA in the same sections (Fig. 3, lower panel). Calbindin-D_28K was used as a marker for the site of active calcium reabsorption in the distal convoluted and connecting tubules of the kidney, where it is co-localized with the sodium-calcium exchanger and calcium pump (48, 50, 51). The colocalization of CaT2 with calbindin-D_28K and NCX1 suggests that CaT2 could participate in apical Ca²⁺ entry in the Ca²⁺ transporting cells of the rat distal nephron as suggested for ECaC in rabbit (27). However, additional transcellular Ca²⁺ pathways may exist in kidney that utilize, for example, the voltage-dependent calcium channels as apical uptake channels for luminal calcium ions (21-24). Also, a capsaicin receptor splice variant was isolated from kidney as a stretch-inhibitable nonselective cation channel (30). Nevertheless, the functional properties of CaT2 also match its postulated role in mediating calcium entry across the apical brush border membrane, as discussed below.

Similar to CaT1 and ECaC, CaT2 mediated saturable, electrogenic calcium uptake when expressed in X. laevis oocytes. The K_m for CaT2-mediated uptake is 0.66 mM (Fig. 6), which is similar to that for CaT1 (0.44 mM) but higher than that for ECaC (0.2 mM). Under voltage clamp (50 mV) and using oocytes preinjected with EGTA, the K_m obtained was much higher (>5 mM) (not shown). The principal differences between the two experimental conditions were (a) the duration of the measurements (a few seconds for voltage clamp, 30 min for uptake), (b) the fact that the membrane voltage was controlled in the voltage clamp but not in the uptake experiments, and (c) the oocytes utilized for voltage-clamp experiments but not those used for measurements of uptake were preinjected with EGTA to a final concentration of 2 mM. Therefore, the K_m obtained by voltage clamp experiments may reflect the initial phase of calcium uptake, while the K_m obtained by uptake experiments may reflect a more integrated measure of uptake occurring during the whole 30-min period. The EGTA preinjection also caused an increase in Ca²⁺ uptake of 3-5-fold (data not shown). It is likely that this was due to the increase in Ca²⁺ concentration gradient across the membrane due to chelation of intracellular Ca²⁺. In addition, chelation of Ca²⁺ may also have attenuated a potential feedback inhibition of CaT2 by Ca²⁺ taken up by the oocytes as suggested by results shown in Fig. 8A. Indeed, calbindin-D_28K could, like EGTA, contribute to removing Ca²⁺ transported by CaT2 into the cytosol immediately beneath the plasma membrane, thereby attenuating this putative feedback inhibition and indirectly enhancing calcium reabsorption. Feedback inhibition (inactivation) by calcium that has been taken up by an influx pathway has been found to be a common mechanism for regulation of voltage-dependent calcium channels (52) and store-operated channels (53, 54) and was also suggested recently to occur for ECaC (55, 56). Further studies of CaT2, however, are needed to identify single channel activity in order to document that it functions as a channel rather than as a facilitated transporter for calcium uptake.

CaT1 and ECaC are rather insensitive to L-type VDCC blockers such as nifedipine, verapamil, and diltiazem (27, 28). Similar results were obtained for CaT2, although we found that the channel blockers, nifedipine, verapamil, and diltiazem, and the L-type calcium channel agonist, Bay K 8644, can exert modest inhibitory effects on CaT2-mediated Ca²⁺ transport, which occur with relatively slow kinetics. These observations may serve as a foundation for developing more specific blockers for CaT2 and similar proteins.

Sodium and chloride had little effect on CaT2-mediated calcium uptake (Fig. 6), a result that was confirmed using voltage clamp measurements (not shown). The sodium conductance was not as significant (not shown) as observed in CaT1-expressing oocytes (28). In contrast, similar to CaT1 (28) and ECaC (27), Ca²⁺ uptake was substantially modulated by extracellular pH, with decreasing activity toward acidic pH and increasing activity toward alkaline pH. The pH modulation of calcium uptake by CaT2 could contribute to the hypercalciuria observed in metabolic acidosis (57-59) and decreased calcium excretion in metabolic alkalosis (60), as suggested previously for ECaC (27).

Similar to CaT1 and ECaC, CaT2 is also permeant to Sr²⁺ and Ba²⁺ but not to Mg²⁺. This suggests that active reabsorption of magnesium in the distal nephron (5) may be mediated by proteins distinct from CaT2. The ratio of the Ca²⁺- to Ba²⁺-evoked currents observed in this study is close to 10:1, suggesting that the Ca²⁺ to Ba²⁺ selectivity of CaT2 differs from that of the PTH-stimulated pathway of calcium entry into distal tubular cells, which has a Ca²⁺ to Ba²⁺ permeability ratio of 16:11 (22). Taken together with the modest dihydropyridine sensitivity exhibited by CaT2, this suggests that the Ca²⁺ influx pathway mediated by CaT2 and the PTH-stimulated dihydropyridine-sensitive pathway studied previously in the distal nephron are distinct.

Besides the alkaline earth metals, Cd²⁺ is also permeable to CaT2. In addition, it is a very potent blocker for CaT2-mediated Ca²⁺ transport. Although Sr²⁺ and Ba²⁺ are also permeable to CaT2, they are not potent blockers of CaT2. From studies of VDCC, low and high affinity binding sites have been proposed as contributing to ion selectivity (61, 62). The dramatic difference between the K_m of Cd²⁺ and its IC₅₀ for inhibition of Ca²⁺ transport suggests that more than one ion binding site exist and that one of them has a very high affinity for Cd²⁺.

Cd²⁺ has been reported to cause a deficit of whole body calcium (63) as well as hypercalciuria (25, 63, 64) and urinary tract and kidney stones (25, 64). Our results provide a possible molecular mechanism(s) for these observations as follows. Dietary Cd²⁺ might be absorbed through the gastrointestinal tract by the metal ion transporter DCT1 (65) and also possibly CaT1 (28); in the kidney, Cd²⁺ might then block Ca²⁺ reabsorption mediated by CaT2, thereby promoting hypercalciuria and kidney stone formation. The deficit in whole body calcium might, therefore, result from Cd²⁺-induced inhibition of gastrointestinal absorption and renal reabsorption of calcium by the human orthologs of CaT1 and CaT2, respectively, or related proteins.

Based on a study of the Cd²⁺ uptake in an immortalized cell line derived from the distal convoluted tubule, Friedman and Gesek (24) demonstrated two pathways of Cd²⁺ uptake: a basal cadmium entry pathway involving carrier-mediated transport that is not dihydropyridine-sensitive and a PTH-stimulated uptake mechanism involving dihydropyridine-sensitive calcium channels. CaT2-mediated Ca²⁺/Cd²⁺ transport is relatively dihydropyridine-insensitive. Moreover, CaT2 functions kinetically as a facilitated transporter despite its channel-like structure. Nevertheless, the relative abilities of CaT2 to transport Cd²⁺ and Ca²⁺ are not entirely dissimilar for the results reported by Friedman and Gesek (24). These properties fit well the basal Cd²⁺ entry pathway, which is dihydropyridine-insensitive. However, further studies are needed to address whether PTH stimulates CaT2-mediated transport of Ca²⁺ and/or Cd²⁺ and whether the dihydropyridine sensitivity of CaT2 is altered after it is stimulated by PTH.

CaT2 is a multitopic protein with six transmembrane domains, a pore region, and ankyrin repeat regions in the amino terminus. While the transmembrane domains and pore region may form a channel for calcium, the ankyrin repeats may serve as the basis for protein-protein interactions with putative molecular partners or could participate in determining CaT2's cellular localization or modulate its function in yet unidentified ways. The multiple protein kinase A and protein kinase C sites may provide the basis for multiple levels of regulation by PTH and other factors regulating distal Ca²⁺ reabsorption. The structure of CaT2 as well as its expression pattern and functional properties are consistent with it mediating calcium entry in rat distal nephron, but further studies of its cellular localization using immunohistochemistry and the use of "knock-out" technologies to prove its functional relevance should provide further insight in this regard.

Voltage-dependent calcium channels have been reported in cells of the distal convoluted tubule (21, 22, 66, 67). Given CaT2's sequence homology and structural similarity to other channels, however, our data (not shown) are not supportive of a close functional relationship between CaT2 and the VDCC, since the latter, unlike CaT2, are very sensitive to nifedipine and/or Bay K 8644 (21, 22, 66, 67). Voltage-dependent calcium channels are known to be composed of multiple subunits including the pore-forming ₁ subunits and cytoplasmic  subunits. Yu et al. (68, 69) reported the presence of multiple isoforms of VDCC ₁ subunits and  subunits in kidney; however, only the _1A and 4 subunits were detected in the distal tubule of the nephron. Barry et al. (23) recently reported that mRNAs encoding the _1C and 3 subunits are present in immortalized DCT cells and that antisense oligonucleotides complementary to the _1C sequence inhibited the rise of intracellular calcium induced by the thiazide diuretic, chlorthiazide, but not that caused by PTH; the antisense sequence to 3, in contrast, inhibited both (23). These results suggested that the _1C subunit may serve as a thiazide diuretic-activated channel, while 3 might serve as a common subunit of both PTH- and diuretic-stimulated calcium channels (23), although the molecular nature of the pore-forming subunit of the PTH-stimulated channel was not defined. The  subunits are known to facilitate the expression and maturation of the pore-forming  subunits (70, 71). It seems likely that CaT2 is a pore-forming subunit of the dihydropyridine-insensitive calcium entry complex; however, determination of whether 3 represents an additional subunit in this complex and whether CaT2 is regulated by PTH require further studies.

In summary, CaT2, a protein closely related to CaT1 and ECaC, is exclusively expressed in the distal nephron of rat kidney, in contrast to CaT1, which is exclusively expressed in rat intestine. CaT2 is relatively insensitive to L-type VDCC antagonists/agonist, although these compounds may bind to the protein with low affinities and slow kinetics. Cd²⁺ is a potent blocker of CaT2-mediated Ca²⁺ transport but is itself permeable through CaT2, albeit with a much lower apparent affinity. This observation provides a plausible explanation for Cd²⁺-induced hypercalciuria and renal stones. It is likely that CaT2 represents one of the proteins mediating the basal level of apical Ca²⁺ entry in the transcellular pathway of calcium reabsorption in the distal convoluted and connecting tubules in rat kidney.

	ACKNOWLEDGEMENT

We are grateful to Dr. Alan S. L. Yu for kindly providing the rat kidney cortex library and the probe for NCX1.

	FOOTNOTES

^* This work was supported by a Brigham and Women's Hospital dual-mentored fellowship (to J.-B. P.) (mentors: M. A. H. and E. M. B.); National Institutes of Health Grants DK41415, DK48330, and DK52005 (to M. A. H. and E. M. B.); and a grant from the St. Giles Foundation (to E. M. B. and P. M. V.). Funding was also supplied by the National Dairy Council (to E. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF209196.

^¶ Recipient of a long term fellowship of the International Human Frontier Science Program.

^** To whom correspondence should be addressed: Harvard Institutes of Medicine, Room 570, 77 Ave. Louis Pasteur, Boston, MA 02115. E-mail: mhediger@rics.bwh.harvard.edu.

Published, JBC Papers in Press, June 29, 2000, DOI 10.1074/jbc.M909686199

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; kb, kilobase pair(s); bp, base pair(s); DIG, digoxigenin; FITC, fluorescein isothiocyanate; VDCC, voltage-dependent calcium channel(s).

	REFERENCES

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