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Localization of the Epithelial Ca²⁺ Channel in Rabbit Kidney and Intestine

JOOST G. J. HOENDEROP^*,, ANITA HARTOG^*, MARCHEL STUIVER^*, ALAIN DOUCET, PETER H. G. M. WILLEMS and RENÉ J. M. BINDELS^*

^* Department of Cell Physiology, Institute of Cellular Signalling, University of Nijmegen, The Netherlands
Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, The Netherlands
Service de Biologie Cellulaire, Centre d'Etudes de Saclay, Gif sur Yvette, France

Correspondence to Dr. René J. M. Bindels, Department of Cell Physiology, University of Nijmegen, Institute of Cellular Signalling, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone : +31 24 3614211 ; Fax : +31 24 3540525 ; E-mail : reneb {at} sci.kun.nl

	Abstract

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Abstract. The epithelial Ca²⁺ channel (ECaC), which is exclusively expressed in 1,25-dihydroxyvitamin D₃-responsive tissues, i.e., kidney, intestine, and placenta, is postulated to constitute the initial step in the process of transcellular Ca²⁺ transport. To strengthen this postulated function, the present study compares the segmental and cellular distribution of ECaC and the other Ca²⁺ transport proteins known to be involved in transcellular Ca²⁺ transport. In rabbit kidney, ECaC mRNA and protein expression were primarily present in the connecting tubule. Immunopositive staining for the ECaC protein was exclusively found at the apical domain of this tubular segment. Importantly, ECaC completely colocalized with calbindin-D_28K, Na⁺-Ca²⁺ exchanger (NCX), and plasma membrane Ca²⁺ -ATPase (PMCA). A minority of cells along the distal tubule lacked immunopositive staining for ECaC and the other Ca²⁺ transporting proteins. These negative cells were identified as intercalated cells. In intestine, ECaC was present in a thin layer along the apical membrane of the duodenal villus tip, whereas the crypt and goblet cells were negative. Again, a complete colocalization was observed between ECaC, calbindin-D_9K, and PMCA. In contrast to the kidney, NCX could not be detected in duodenum. The present finding that ECaC completely colocalizes with the Ca²⁺ transport proteins in the connecting tubule and duodenum, together with its apical localization, further substantiates the postulated function of ECaC as the gatekeeper of active Ca²⁺ (re)absorption.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, the epithelial Ca²⁺ channel (ECaC) was cloned from rabbit kidney and implicated to play a crucial role in Ca²⁺ homeostasis. Functional and molecular biologic analysis revealed that ECaC has all the defining properties for being the gatekeeper of active transcellular Ca²⁺ transport in 1,25-dihydroxyvitamin D₃-responsive epithelia, i.e., kidney, small intestine, and placenta (1,2,3).

The gastrointestinal tract and kidney determine the net intake and output of Ca²⁺ for the entire body, and thereby the overall state of Ca²⁺ balance. The most important underlying mechanism is 1,25-dihydroxyvitamin D₃-regulated active transport of Ca²⁺ from the lumen to the blood compartment, which occurs primarily in the proximal small intestine (4) and the distal part of the nephron (5). This process of transcellular Ca²⁺ transport is currently envisioned as a three-step operation consisting of passive apical Ca²⁺ entry via ECaC followed by cytosolic diffusion facilitated by Ca²⁺ binding proteins, calbindin-D_28K in kidney and calbindin-D_9K in intestine, and active extrusion across the opposing basolateral membrane by a highaffinity Ca²⁺-ATPase (PMCA) and/or a Na⁺-Ca²⁺ exchanger (NCX) (2, 4, 5). From an energetic point of view, it is likely that the initial apical influx of Ca²⁺ forms the rate-limiting step in this process and, therefore, the final regulatory target for stimulatory and inhibitory hormones (2).

The aim of the present study was to further substantiate the postulated function of ECaC as the Ca²⁺ entry mechanism initiating active Ca²⁺ transport. To this end, we have investigated ECaC mRNA expression in microdissected rabbit nephron segments. In addition, the (sub)cellular localization of ECaC with respect to the other Ca²⁺ transport proteins was studied in rabbit kidney and intestine by immunohistochemistry.

	Materials and Methods

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Isolation of Nephron Segments and Total RNA Extraction
Rabbit nephron segments were obtained by microdissection as described previously (6). Briefly, microdissection medium was prepared from Hanks' sterile solution (MEM 2126 ; Eurobio, Les Ulis, Cedex, France ; 137 mM NaCl, 5 mM KCl, 0.8 mM MgSO₄, 0.33 mM Na₂HPO4, 0.44 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂) supplemented with 1 mM lactate, 1 mM pyruvate, 1 mM glutamine, 1 mg/ml protease-free bovine serum albumin, and 20 mM Hepes, and pH was adjusted to 7.4. After anesthesia (Nembutal, 5 mg/100 g body wt), the left kidney was perfused with collagenase dissolved in microdissection medium (1.6 mg/ml). Thin pyramids, cut along the corticomedullary axis, were incubated for 25 min at 30°C in microdissection solution containing collagenase (1.2 mg/ml). The following structures were isolated according to morphologic and topographic criteria : glomeruli, proximal convoluted and straight tubules (PCT and PST), medullary and cortical thick ascending limbs of Henle's loop (mTAL, cTAL), distal convoluted tubules (DCT), cortical connecting tubules (CNT), and cortical collecting ducts (CCD). After elimination of cells and debris, pools of 10 to 50 tubules were transferred with 5 ml of dissection medium and processed for RNA extraction. Total RNA was extracted from isolated nephron segments and total kidney cortex according to Chomczynski and Sacchi and downscaled for small amounts (6, 7).

Reverse Transcription-PCR of Rabbit ECaC RNA
ECaC sense primer (5'-TGAACCTGGTGCGCGCACTGC-3') and ECaC antisense primer (5'-CCCAGGGAGTCCTGGGCCCGG-3') used in reverse transcription-PCR experiments were designed according to the rabbit ECaC cDNA sequence (GenBank accession no. AJ133128) and cover nucleotide positions 488 to 507 and 665 to 685 of the cDNA, respectively (1). The ß-actin mRNA level was determined as a control to demonstrate the integrity of the isolated RNA from the nephron structures by using primers corresponding to the coding region of the human ß-actin cDNA. The ß-actin sense and antisense primers were : 5'-GCTACGAGCTGCCTGACGG-3' and 5'-GAGGCCAGGATGGAGCC-3', respectively, and bracket the sequence from 757 to 1084 bp of the human ß-actin (8). Briefly, in a final volume of 34 µ1 the following compounds were added and maintained on ice : total RNA (approximately 20 ng) of isolated nephron structures and antisense primer (10 pmol). For denaturation, the tubes were heated at 70°C for 5 min and then equilibrated at 37°C. Each sample was separated into two incubations of 17 µ1 and supplemented with 12 µ1 of reverse transcriptase mix containing 6 µ1 of 5x first-strand buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl₂), 2 µ1 of 10 mM dNTP, 3 µ1 of 0.1 M dithiothreitol, and 1 µ1 of RNasin (40 U). The samples were incubated for 15 min at 37°C, and subsequently one reaction was supplemented with 200 U of Moloney murine leukemia virus-reverse transcriptase (Life Technologies, Breda, The Netherlands), whereas another reaction was used as a control for DNA contamination. The reverse transcription (RT) reaction was performed at 37°C for 1 h, followed by an increase to 90°C for 2 min. The amplification reaction was initiated by adding to each sample 50 µ1 of PCR mix containing 2 mM MgCl₂, 10 pmol of the sense primer, 0.01 mg/ml gelatin, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1.25 U Taq polymerase. The PCR was based on 40 cycles of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C, with an elongation time of 10 min being used after the last cycle. After the RT-PCR amplification step, an aliquot of 20 µ1 from each sample was run on a 2% agarose gel containing 2.5 µg/ml ethidium bromide, and bands were visualized using ultraviolet illumination.

Generation and Characterization of Antisera
Antiserum against ECaC was obtained by immunization of guinea pigs with ECaC C-tail protein and affinity-purified as described previously (1). To demonstrate the specificity of the ECaC antibody, immunoblots using a membrane fraction of ECaC-expressing oocytes and rabbit kidney cortex, and isolated rabbit duodenal brush-border membranes were performed. Unfortunately, we were unable to demonstrate a specific positive band of the appropriate molecular mass. Subsequently, we have generated two series of antibodies against a 15 AA synthetic peptide representing the C-tail of ECaC and against a fusion protein of the extracellular loop between transmembrane segment 1 and 2, respectively. Immunohistochemistry performed with these new antibodies demonstrated identical localization of ECaC in rabbit kidney cortex and duodenum as obtained in the present study. As with our first antibody, these new antibodies did not produce a specific signal on immunoblots, suggesting that the expression level of ECaC might be too low to detect the protein by immunoblots. Thus, three antibodies directed against different parts of the ECaC protein independently illustrated identical localization of ECaC. This convincingly demonstrates the specificity of the observed immunohistochemistry. Furthermore, Xenopus laevis oocytes were injected with 2 ng of ECaC cRNA. After 2 d, oocytes were fixed and stained for ECaC. Immunopositive staining was exclusively present in the plasma membrane of injected oocytes, whereas noninjected oocytes were devoid of any positive staining as shown previously (3). As an additional control, COS cells were transfected with rabbit ECaC cDNA and after 1 d cells were fixed and stained for ECaC. ECaC protein was observed in the plasma membrane of transfected COS cells, whereas nontransfected cells were negative (data not shown). Rabbit antisera against rabbit calbindin-D_28K and bovine calbindin-D_9K were characterized previously (1, 9). Mouse anti-sarcolemmal Na⁺-Ca²⁺-exchanger monoclonal antibody (mAb) (C2C12) was a generous gift from Dr. K. D. Philipson (Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, University of California, Los Angeles, School of Medicine, Los Angeles, CA), mouse anti-plasma membrane Ca²⁺-ATPase mAb (5F10) was kindly provided by Dr. J. T. Penniston (Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN), and the mouse monoclonal antibody against the thiazide-sensitive NaCl cotransporter (TSC) (JM5) was a gift from Dr. D. H. Ellison (Department of Medicine, University of Colorado School of Medicine, Denver, CO). The characterization of these mAb has been described elsewhere (10,11,12,13).

Immunohistochemistry
Kidneys and intestine were obtained from New Zealand White rabbits (approximately 0.5 kg). The organs were cut into pieces, placed in 1% (wt/vol) periodate-lysine-paraformaldehyde fixative (9) for 2 h, and overnight incubated in phosphate-buffered saline (PBS) containing 15% (wt/vol) sucrose. Subsequently, samples were frozen in liquid nitrogen and 7-µm sections were used for different staining procedures. Duodenum sections were boiled for 2 min, cooled to room temperature in citrate buffer (pH 6) containing 0.01 M C₆H₅Na₃O₇ and 0.01 M C₆H₈O₇, and thoroughly rinsed with PBS.

For immunoperoxidase staining, kidney and intestine sections were incubated for 30 min at room temperature in PBS containing 0.3% (vol/vol) H₂O₂ and washed three times with TN buffer (0.15 M NaCl, 0.1 M Tris/HCl, pH 7.5). Subsequently, sections were incubated for 30 min in TNB buffer (TN buffer containing 0.5% [wt/vol] blocking reagent from NEN Life Science Products, Zaventem, Belgium). Sections were incubated for 16 h at 4°C in TNB buffer containing the guinea pig anti-ECaC antiserum (1 : 50). Sections were washed three times with TNT buffer (TN buffer containing 0.05% [vol/vol] Tween 20) and incubated for 30 min at room temperature with peroxidase-conjugated goat anti-guinea pig antibody (1 : 50) (Sigma, St. Louis, MO). The sections were counterstained with hematoxylin Gill 2 and embedded in glycerol/gelatin as described previously (9).

For double immunofluorescence staining, periodate-lysine-paraformaldehyde-fixed frozen sections were preincubated in TNB buffer for 30 min and incubated for 16 h at 4°C with antiserum against ECaC (1 : 200) and with one of the antibodies against the Ca²⁺ transport proteins (calbindin-D_28K 1 : 200, calbindin-D_9K 1 : 100, NCX 1 : 100, or PMCA 1 : 400). Sections double-stained for ECaC and TSC were preincubated in TNB buffer for 30 min and incubated for 16 h at 4°C with antiserum against ECaC (1 : 200), subsequently washed with TNT buffer, and incubated for 1 h at room temperature with antiserum against TSC (1 : 3200).

After thorough washing with TNT buffer, tissue sections stained for ECaC together with NCX, PMCA, or TSC were incubated at room temperature for 1 h with goat anti-guinea pig-Alexa 594-conjugated anti-IgG (1 : 300) (Molecular Probes, Eugene, OR) for the detection of ECaC and with goat anti-mouse-Alexa 488-conjugated anti-IgG (1 : 300) (Molecular Probes) for the detection of the other transporters. Double staining of ECaC and calbindins was performed by incubation of the tissue sections for 1 h at room temperature with goat anti-guinea pig-FITC-conjugated anti-IgG (1 : 300) (Sigma) for the detection of ECaC and with goat anti-rabbit tetramethylrhodamine isothiocyanate-conjugated anti-IgG (1 : 300) (Sigma) for the detection of calbindin-D_28K and calbindin-D_9K, respectively. Kidney sections were incubated for 16 h at 4°C with FITC-conjugated peanut lectin (1 : 200) to visualize intercalated cells.

All sections were rinsed twice with TNB buffer and mounted in mowiol (Hoechst, Frankfurt, Germany) containing 2.5% (wt/vol) NaN₃ as described previously (1). All negative controls, including sections incubated with either preimmune serum, preabsorbed antiserum for 1 h with 10 µg/ml ECaC-glutathione S-transferase fusion protein, or solely with conjugated secondary antibodies, were devoid of any staining. Photographs were taken with a Zeiss Axioskop microscope equipped for epifluorescence illumination using Kodak EPH P1600X film or with a Bio-Rad MRC 1000 confocal laser scanning microscope.

	Results

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Segmental Distribution of ECaC mRNA
RT-PCR on total RNA isolated from rabbit kidney cortex yielded a single amplified fragment of expected size of 197 bp (Figure 1). This band was absent when the RT step was omitted, which excludes amplification of contaminating genomic DNA (not shown). Sequence analysis confirmed that the amplified fragment encoded the part of the amino-terminal domain of rabbit ECaC between nucleotide positions 488 and 685. Subsequently, duplicate RT-PCR assays were performed on each of two batches of segmental RNA isolated from two rabbits. The acquired results demonstrate that the ECaC transcript is exclusively present in distal tubular segments. The expression was highest in CNT and decreased in the flanking DCT and CCD. No signal was observed in glomeruli, proximal tubules, thick ascending limb of Henle, or the outer medullary collecting duct. The integrity of isolated RNA was demonstrated by the presence of ß-actin-specific fragments of the correct size (Figure 1). PCR water controls were consistently negative.

View larger version (46K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Reverse transcription-PCR analysis of epithelial Ca2+ channel (ECaC) mRNA expression in renal tubules. Total RNA was isolated from the indicated nephron structures microdissected from rabbit kidney, including glomeruli (GM), proximal convoluted and straight tubules (PCT and PST), medullary and cortical thick ascending limbs of Henle's loop (mTAL and cTAL), distal convoluted tubules (DCT), connecting tubules (CNT), and cortical and outer medullary collecting ducts (CCD and OMCD). Rabbit kidney cortex total RNA was used as a positive control. The size of the expected amplified rabbit ECaC fragment (197 bp) is indicated. ß-actin mRNA was used as a control to demonstrate the intactness of isolated RNA. As a negative control, water was added instead of cDNA.

Immunohistochemical Localization of ECaC
The localization of ECaC was further investigated using an affinity-purified antibody raised against the carboxy terminus of ECaC as described previously (1, 3). The distribution of ECaC-specific immunoperoxidase staining in rabbit kidney cortex and duodenum is shown in Figure 2, A through F. In kidney, ECaC-positive staining was found in the superficial cortex, whereas outer and inner medulla were negative. Importantly, the exclusive cortical staining for ECaC was predominantly found along the apical membrane of the distal tubular segments (Figure 2, A through C). Staining was not observed in glomeruli, proximal tubular segments, or the loop of Henle. In intestine, ECaC was abundantly present in brush-border membranes of duodenum (Figure 2, D and E). Absorptive epithelial cells in the villi stained intensely for ECaC (Figure 2E), whereas crypt cells were negative (Figure 2F). Immunopositive staining for ECaC was not observed in ileum.

View larger version (127K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Immunoperoxidase staining for ECaC of periodate-lysine-paraformaldehyde-fixed sections of rabbit kidney cortex and duodenum. In kidney, apical localization of ECaC was observed in distal tubular segments (A through C). Sections of outer and inner medulla were negative (not shown). In duodenum, intense immunoperoxidase staining was observed in absorptive cells lining the villus tip (D and E), whereas crypt cells showed little or no staining (F). Ileum was negative (not shown). Bars : 25 µm in A through C ; 100 µm in D ; and 50 µm in E and F.

To determine more precisely the distribution of the ECaC protein along the distal nephron, several criteria were used. First, tubules were identified by their morphologic and anatomical features (14). Second, colocalization studies were performed with antisera against TSC, as a marker for DCT (13), and the Ca²⁺ transport proteins. Third, CNT and CCD were recognized by the appearance of intermingled intercalated cells. Discrimination between these segments was based on a different position in the cortex and superficial T branches, indicating transitions between these latter segments (15). Figure 3 shows double labeling of rabbit kidney cortex section for the presence of ECaC (Panel A) and TSC (Panel B) depicting immunopositive distal tubular segments. Some distal segments stained intensely for TSC, but were negative for ECaC. However, other tubules stained lightly for TSC and were also positive for ECaC. Because the former TSC-positive segments were previously identified as DCT and the latter as CNT (13), these findings suggest that ECaC is present in CNT, but not in DCT. The staining of ECaC did extend into CCD, as superficial T-branched tubules positive for ECaC were identified (Figure 3C). Immunopositive staining was not present in that part of the CCD located in medullary rays. A minority of the cells within the ECaC-positive distal tubules did not express ECaC, indicating that they are likely intercalated cells. Indeed, cells lacking ECaC were identified as intercalated cells using double fluorescence staining with peanut lectin (Figure 3D). The kidney section incubated with preabsorbed antiserum for 1 h with 10 µg/ml ECaC-glutathione S-transferase fusion protein was devoid of any staining (Figure 3E). Thus, ECaC is predominantly expressed in CNT, which is in agreement with the fact that this nephron segment is in rabbit the primary segment of hormone-regulated Ca²⁺ reabsorption (2, 5).

View larger version (60K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Distribution of ECaC along the distal nephron. Double immunofluorescence staining of rabbit kidney cortex sections showing staining for ECaC (A) and the thiazide-sensitive NaCl cotransporter (TSC) (B). The asterisks indicate DCT cells intensely stained for TSC but negative for ECaC, while the arrowheads mark CNT cells lightly positive for TSC and strongly stained for ECaC. G, glomerulus. (C) A superficial T-branched tubule positive for ECaC. (D) A superimposed picture of a CNT with immunopositive staining for ECaC (green) and peanut lectin (red). (E) The absence of immunopositive staining with ECaC antiserum preabsorbed for 1 h with 10 µg/ml ECaC-glutathione S-transferase fusion protein. G, glomerulus. Bars : 30 µm in A and B ; 15 µm in C and E ; and 8 µm in D.

ECaC Colocalizes with the Ca²⁺ Transport Proteins in Kidney and Intestine
Double immunofluorescence labeling experiments were performed to investigate the localization of ECaC with respect to the other Ca²⁺ transport proteins involved in transcellular Ca²⁺ transport. The antibodies used were raised against the canine sarcolemmal NCX, human erythrocyte PMCA, rabbit kidney calbindin-D_28K, and bovine intestinal calbindin-D_9K. Double staining of kidney sections revealed consistent colocalization of ECaC with calbindin-D_28K, NCX, and PMCA in the distal nephron (Figure 4). PMCA was also found in TAL (not shown). Immunopositive staining for NCX was restricted to the basolateral membrane of distal tubular cells, whereas staining for both calbindin-D_28K and PMCA was observed throughout the whole cell. In line with the kidney, a complete colocalization between ECaC, calbindin-D_9K, and PMCA was observed in duodenum (Figure 5). PMCA was present along the lateral membrane, whereas calbindin-D_9K was found throughout the whole cell. NCX could not be detected in duodenum.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Double immunofluorescence staining of rabbit kidney cortex sections showing staining for ECaC in distal tubular segments (A, C, and E) and calbindin-D28K (B), Na+-Ca2+ exchanger (D), and PMCA (F). Bar, 30 µm.

View larger version (87K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Immunofluorescence staining of serial sections of rabbit duodenum showing staining for ECaC (A), calbindin-D9K (B), and PMCA (C). Bar, 40 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that in both kidney and intestine, ECaC colocalizes with the other Ca²⁺ transporting proteins known to be involved in transcellular Ca²⁺ transport, confirming that ECaC is indeed the gatekeeper in transepithelial Ca²⁺ transport.

In rabbit, CNT has been implicated as the primary segment of hormone-regulated Ca²⁺ reabsorption (2,5), whereas in most other mammalian species including humans, this hormone-dependent regulation is situated mainly in DCT (2,5,16). Therefore, the present findings that ECaC staining is absent in DCT and intense in CNT and completely colocalizes with the other Ca²⁺ transport proteins, together with previous studies documenting calbindin-D_28K and NCX expression exclusively in rabbit CNT and to a lesser extent in CCD (17,18,19), strengthens the idea that ECaC is involved in active Ca²⁺ transport. ECaC was predominantly localized to the apical membrane, which is in accordance with its postulated function as the Ca²⁺ entry mechanism. Calbindin-D_28K was uniformly distributed throughout the cytosol, which is in line with the physiologic role assigned to this Ca²⁺ transport protein. It has been postulated that calbindin-D_28K acts both as a cytosolic Ca²⁺ buffer and as a shuttle mechanism between the luminal influx and basolateral efflux sites (20, 21). NCX was exclusively present in a layer along the basolateral membrane, whereas PMCA was present throughout the whole cell.

ECaC mRNA was faintly detected in microdissected rabbit DCT, whereas these tubule segments did not stain for ECaC protein. One explanation for this discrepancy is that during the microdissection procedure, ECaC-containing CNT cells were coisolated. Alternatively, the protein level of ECaC in DCT may be too low to be detected by immunohistochemistry.

It has been reported that the proximal tubule and thick ascending limb of Henle's loop exhibit, besides paracellular Ca²⁺ reabsorption, a residual portion of active Ca²⁺ transport (5, 22, 23). However, the consistent absence of the Ca²⁺ transport proteins is not in line with the occurrence of active Ca²⁺ reabsorption in these segments.

In terms of distal Ca²⁺ reabsorption, it is interesting to note that thiazide diuretics have been demonstrated to dissociate Na⁺ and Ca²⁺ transport by inhibiting Na⁺ uptake via TSC and promoting Ca²⁺ reabsorption (24). Likewise, loss of function of TSC in patients suffering from Gitelman's syndrome is associated with severe hypocalciuria (25). The proposed explanation for the diminished Ca²⁺ excretion is that impairment of apical NaCl entry hyperpolarizes the cell and lowers the intracellular Na⁺ concentration, stimulating the entry of Ca²⁺ through the apical Ca²⁺ channel and facilitating the removal of Ca²⁺ via the exchange of Na⁺ for Ca²⁺ across the basolateral membrane, respectively (25, 26). The specific localization of ECaC shown here warrants additional studies addressing the above-mentioned mechanism by cell physiologic approaches. Electrophysiologic characterization of ECaC expression in Xenopus laevis oocytes has already revealed that Ca²⁺ influx via ECaC is indeed stimulated by hyperpolarizing membrane voltages, which supports this concept (3).

Previous studies demonstrated that CNT and CCD are comprised of mainly principal and some intercalated cells, which are known to display distinct transport processes (5, 9, 27). Confocal fluorescence microscopy showed that within the ECaC-positive distal tubular segments, a minority of cells were devoid of Ca²⁺ transport proteins, and labeling with peanut lectin identified these negative cells as intercalated cells. This specific localization of ECaC in the Ca²⁺-transporting principal cells is completely in agreement with its postulated function in Ca²⁺ reabsorption.

In intestine, ECaC is exclusively localized in the brushborder membrane of duodenum and could not be detected in ileum. This is in line with our previous finding that ECaC mRNA is highly expressed in duodenum, decreased in jejunum, and absent in ileum and colon (1). Wasserman and Fullmer (4) demonstrated that 1,25-dihydroxyvitamin-D₃-regulated Ca²⁺ absorption occurs primarily in the absorptive cells of duodenum. In this context, the present finding that ECaC lines the duodenal villus tip, while it is not expressed in crypt and goblet cells, supports the idea that this Ca²⁺ channel is the Ca²⁺ entry mechanism. As in kidney, a complete colocalization of ECaC with the Ca²⁺ transport proteins was observed in the proximal part of the intestine. Of note, NCX was not detected in small intestine. This is in agreement with previous studies showing that Na⁺-Ca²⁺ exchange is of minor importance in the process of intestinal Ca²⁺ absorption (28). It is therefore likely that immunodetection of NCX in duodenum is difficult due to a low expression level. Alternatively, another isoform of NCX could be present that is not detected by the antibody used in the present study.

In conclusion, the present data further substantiate the postulated function of ECaC as the gatekeeper of active Ca²⁺ (re)absorption. First, ECaC is present in the segments implicated in Ca²⁺(re)absorption in rabbit kidney and intestine. Second, ECaC is consistently localized to the apical membrane. Third, ECaC colocalizes with the vitamin D₃-dependent calbindins, Na⁺-Ca²⁺-exchanger, and Ca²⁺-ATPase and completes the family of Ca²⁺ transport proteins involved in transepithelial Ca²⁺ transport in kidney and intestine.

	Acknowledgments

 
This study was supported in part by a grant from the Dutch Organization of Scientific Research (NWO-ALW 805-09.042). We thank Drs. J. T. Penningston, K. D. Philipson, and D. H. Ellison for kindly providing antibodies 5F10, C2C12, and JM5, respectively.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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