The alpha 1G-subunit of a voltage-dependent Ca2+ channel is localized in rat distal nephron and collecting duct

Ditte Andreasen1, Boye L. Jensen1, Pernille B. Hansen1, Tae-Hwan Kwon2, Søren Nielsen2, and Ole Skøtt1

1 Department of Physiology and Pharmacology, University of Southern Denmark-Odense University, DK-5000 Odense; and 2 Department of Cell Biology, University of Aarhus, DK-8000 Aarhus, Denmark


    ABSTRACT
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The molecular type and localization of calcium channels along the nephron are not well understood. In the present study, we assessed the distribution of the recently identified alpha 1G-subunit encoding a voltage-dependent calcium channel with T-type characteristics. Using a RNase protection assay, alpha 1G-mRNA levels in kidney regions were determined as inner medulla outer medulla congruent  cortex. RT-PCR analysis of microdissected rat nephron segments revealed alpha 1G expression in the distal convoluted tubule (DCT), in the connecting tubule and cortical collecting duct (CT+CCD), and inner medullary collecting duct (IMCD). alpha 1G mRNA was expressed in the IMCD cell line mIMCD-3. Single- and double-labeling immunohistochemistry and confocal laser microscopy on semithin paraffin sections of rat kidneys by using an anti-alpha 1G antibody demonstrated a distinct labeling at the apical plasma membrane domains of DCT cells, CT principal cells, and IMCD principal cells.

distal convoluted tubule; inner medullary collecting duct; voltage-operated calcium channel


    INTRODUCTION
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INTRODUCTION
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TRANSMEMBRANE MOVEMENTS OF calcium are of importance for a wide range of biological processes in the kidney. Influx of extracellular calcium is required for signal transduction in renal vascular smooth muscle cells and in various tubular epithelial cells. Furthermore, the maintenance of whole body calcium homeostasis requires uptake of filtered calcium ions across the apical membranes of certain renal epithelial cells.

Voltage-gated calcium channels comprise a group of transmembrane multisubunit proteins (3) with a central pore-forming alpha 1-subunit containing the voltage sensor and drug receptor. Primary distinction between classes of voltage-gated calcium channels is between high-voltage-activated, comprising L-, N-, P/Q-, and R-type channels, and low-voltage-activated, comprising only T-type channels (for an overview, see 16). Yu et al. (32) demonstrated expression of at least four alpha 1-subunit genes in the kidney (alpha 1A, alpha 1C, alpha 1D, and alpha 1S), all of which encode high-voltage activated calcium channels. Recently, the first gene encoding an alpha 1-subunit with T-type characteristics, alpha 1G, was cloned and found to be expressed in whole kidney by Northern blotting (17, 21). We considered it relevant to examine the nephron localization of the alpha 1G gene product, and to this end we employed several methods. Semiquantitative estimation of the mRNA abundance in kidney regions was done by RNase protection assay, exact localization of mRNA expression in the renal tubules was obtained by RT-PCR analysis on microdissected tubular segments, and, finally, the alpha 1G-subunit protein was localized by immunohistochemistry in semithin cryosections.

The results show that the alpha 1G-subunit is expressed in distinct nephron segments. We found alpha 1G mRNA in the distal part of the nephron as well as in the inner medullary collecting duct. These same segments also were labeled at the apical plasma membrane domains by using an antibody specific for alpha 1G.


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In vivo protocols. Male Sprague-Dawley rats (150-200 g), with free access to standard rodent diet (Altromin C1000, Lage, Germany) and tap water, were used for the experiments. All animal procedures conformed with the Danish law on experiments using animals as well as the guidelines for the care and handling of animals established by the US Department of Health and Human Services and published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).

Cell culture. The mIMCD-3 cell line (22) was obtained from the American Tissue Type Culture Collection (CRL-2123) and maintained according to instructions. In brief, the cells were grown in a 1:1 ratio of DMEM and Ham's F-12 medium, with addition of 10% fetal bovine serum. Medium was renewed two to three times weekly. Twenty hours before harvest, serum was removed. The cells were washed with PBS, and RNA was isolated by using the RNeasy kit according to manufacturer's instructions (Qiagen, KeboLab).

Microdissection of rat nephron segments. Nephron segments were obtained by microdissection of rat kidney tissue from a total of 10 male Sprague-Dawley rats according to the protocol of Yang et al. (30). In brief, rats were anesthetized by an intraperitoneal injection of mebumal, and the aorta was cannulated. Both kidneys were perfused after the aorta had been ligated, and the left renal vein was opened. First, kidneys were perfused with 20 ml of cold isotonic saline followed by 20 ml of DMEM with 0.1% albumin and collagenase A (1 mg/ml-0.2 U/mg, Roche Molecular Biochemicals). The kidneys were removed and placed in cold saline. Thin coronal slices were cut with a razor blade, and the slices were digested further in 25 ml DMEM with 0.1% BSA and 1 mg/ml collagenase A for 25 min at 37°C during modest shaking. Next, the tissue was washed twice with DMEM containing 10% FCS and kept on ice in this medium. Microdissection was done under a stereomicroscope with sharpened forceps. The nephron segments were identified by their localization in kidney regions and by their thickness and appearance. We obtained segments of the proximal convoluted tubule (PCT), proximal straight tubule (PST), descending thin limb of Henle's loop (DTL), thin limb of Henle's loop from inner medulla (TL), medullary and cortical thick ascending limb of Henle's loop (mTALH and cTALH, respectively), distal convoluted tubule (DCT), connecting tubule and cortical collecting duct (CT+CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). To rinse off any contaminating loose cells arising from the dissection, the isolated segments were transferred in 3-5 µl DMEM to 500 µl fresh DMEM in a 24-well cell culture plate on ice. Identical segments were pooled, and the total length of tubule was assessed by using a calibrated micrometer scale built into the ocular. The segments were transferred to 400 µl guanidinium thiocyanate (4 M) solution, and 12 µg yeast tRNA were added as carrier. Samples were stored at -80°C until RNA extraction. Finally, RNA was isolated by a modified phenol-chloroform extraction protocol (6).

RT-PCR and cloning. RT-PCR was performed as described previously (15). PCR primers for alpha 1G (DNA Technology, Aarhus, Denmark) were rat, forward 5'GAA CGT GAG GCC AAG AGT '3, reverse 5'GCT TGT ATG CGT TCC CCT '3, covering bases 3910-4130, 221 bp (GenBank accession no. AF027984); and mouse, forward 5' CGG GAT CCT GGC AAG TCG '3, reverse 5' CGG AAT TCG ATG ATC CGG '3, covering bases 3711-3930, 220 bp (GenBank accession no. AJ012569). Linker sequences (8 bp each) were added to introduce EcoR I and BamH I restriction sites for cloning. beta -Actin primers were copied from Yu et al. (32). For nephron distribution, cDNA equivalent to 1 (alpha 1G)- or 0.2-mm tubule (actin) was used. Negative controls included water instead of cDNA in the PCR, and carrier tRNA with no further addition of RNA in the RT and subsequent PCR. The rat alpha 1G amplification product was cloned in vector pSP73 (Promega) by standard methods (24). The insert was sequenced by using SP6- and T7-specific primers. Mouse PCR products were directly sequenced, using PCR primers.

Southern blotting. DNA probes were synthesized with 1 µCi/µl [alpha -32P]dCTP (Amersham Pharmacia Biotech) and 0.05 U/µl Klenow by standard methods (24). DNA was transferred by capillary blotting (24) to Zeta Probe GT membrane (Bio-Rad, Copenhagen, Denmark), and hybridization of radioactive probe was allowed overnight at 42°C (24). The membrane was washed, and autoradiography was performed for 2-4 h.

RNase protection assays. Animals were killed by decapitation, and organs were rapidly removed, frozen in liquid nitrogen, and stored at -80°C. The kidneys were separated into major regions by dissection under a stereomicroscope and frozen in liquid nitrogen. The medullary rays were contained in the cortical tissue, and no attempt was made to separate the outer and inner stripe of the outer medulla. RNA was extracted according to the acid-guanidinium-phenol-chloroform extraction protocol (6).

mRNA levels for alpha 1G, renin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed by A/T1 RNase protection assay as described (11, 15, 26). Plasmids carrying a 296-bp fragment of rat preprorenin and a 342-bp fragment of rat GAPDH have previously been constructed (15, 26). After linearization with Hind III, the plasmids yielded radiolabeled antisense transcripts by incubation with SP6 polymerase (Promega) and [alpha -32P]GTP (Amersham) according to the Promega riboprobe in vitro transcription protocol. For hybridization, 5 × 105 counts/min of the probe were used with samples of total RNA at 60°C overnight in a final volume of 50 µl. Sequential digestions were performed with a mixture of RNase A/T1 (Roche Molecular Biochemicals) and proteinase K (Roche Molecular Biochemicals). Protected probes were separated by gel electrophoresis on 8% polyacrylamide gels. Autoradiography was done at -80°C for 1-3 days. Radioactivity from the protected probe was determined in a beta scintillation counter.

Antibody against the alpha 1G-subunit of a voltage-dependent calcium channel. Rabbit polyclonal antisera against rat alpha 1G were raised by using synthetic peptides corresponding to amino acids 1-22 of the NH2 terminal of rat alpha 1G-subunit of a voltage-dependent T-type calcium channel (GenBank accession no. AF027984) with addition of a COOH-terminal cysteine. Analysis using the BLAST program (National Library of Medicine, Bethesda, MD) revealed no homology of the immunizing peptide with any known proteins. The purified peptide was conjugated to keyhole limpet hemocyanin for immunization in rabbits. For immunolocalization, the antisera were affinity purified against immunizing peptides by using Immunobilization Kit No. 2 (Pierce, Rockford, IL).

Immunoperoxidase microscopy and laser confocal microscopy for alpha 1G-subunit of a voltage-dependent calcium channel. Kidneys from Munich-Wistar rats were fixed by retrograde perfusion via the aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer (31). Semithin paraffin sections (2-3 µm) were incubated overnight at 4°C with anti-alpha 1G (diluted 1:8), and labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P448, 1:100, DAKO, Glostrup, Denmark). Immunolabeling controls were performed by using preabsorption of antibodies with the alpha 1G-immunizing peptides. As a control on which cell types labeled positive in the distal convoluted tubule and connecting tubule, we also labeled adjacent serial sections with anti-alpha 1G, anti-calbindin D-28K monoclonal mouse antibodies (Research Diagnostics, Flanders, NJ) and polyclonal affinity-purified rabbit anti-H+-ATPase (beta 1-subunit) antibodies (kind gift from Dr. Mark Knepper, NIH). For double-labeling fluorescence microscopy, the vacuolar H+-ATPase was localized by using a mouse monoclonal antibody (E11, diluted 1:100, kindly supplied by Dr. S. Gluck), which was mixed with the primary antibodies against anti-alpha 1G (diluted 1:8). The labeling was visualized by using both Alexa 546-conjugated goat anti-mouse antibody and Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes). The microscopy was carried out by using a Leica DMRE light microscope and a Zeiss LSM510 laser confocal microscope.


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Validation of assays. The rat alpha 1G PCR product had the expected size of 237 bp (Fig. 3A), and the sequence was identical to the published rat alpha 1G cDNA. The mouse alpha 1G PCR product had the expected size of 236 bp (Fig. 3B), and the sequence was identical to the recently published mouse sequence (17), which is 91% homologous to the rat sequence. A 32P-labeled antisense riboprobe against the rat sequence was used to assay for the alpha 1G-subunit mRNA in samples of total RNA from rat kidney regions. The alpha 1G mRNA assay was linear at least in 10- to 80-µg range whole kidney total RNA and resulted in hybrids of the expected sizes, confirming renal expression of this subunit (not shown).

Distribution of alpha 1G mRNA in renal regions. RNase protection assays were performed on RNA extracted from the renal regions (cortex, outer medulla, and inner medulla). alpha 1G mRNA was expressed in all regions of the kidney (Fig. 1A). The housekeeping gene GAPDH was not differentially expressed in kidney regions (cortex 311 ± 28 cpm, n = 6; outer medulla 353 ± 50 cpm, n = 6 ; and inner medulla 226 ± 33 cpm, n = 6, nonsignificant by ANOVA, where cpm is counts/min). Comparisons between regions were made with GAPDH-normalized values. Thus the alpha 1G-subunit was 40-fold more abundant in the inner medulla compared with cortex and outer medulla, where levels were close to equal (Fig. 1B). By the same method, renin mRNA was detected in the kidney cortex at levels 25-fold higher compared with outer medulla (Fig. 1B,) and in the inner medulla no renin signal was recorded. This confirms an accurate separation of kidney regions.


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Fig. 1.   Distribution of alpha 1G-subunit mRNA in rat kidney regions. A: autoradiographs of RNase protection assays on kidney regions: cortex, outer medulla (OM), and inner medulla (IM). Top: probe specific for alpha 1G mRNA. Bottom: probe specific for glyceraldehyde-3-phopshate dehydrogenase (GAPDH) mRNA. B: GAPDH-normalized values of protected alpha 1G and preprorenin mRNA levels in kidney regions. cpm, Counts/min.

Tissue distribution for the alpha 1G-subunit. It has previously been reported that alpha 1G is predominantly a neuronal isoform and weakly expressed in lung, kidney, and skeletal muscle (17, 21). By means of an RNase protection assay, these tissues were compared with the kidney inner medulla for alpha 1G expression. alpha 1G was expressed in the order brain > inner medulla > heart > lung skeletal muscle (Fig. 2A). Except for the inner medulla, which has not previously been assayed, this is in accordance with published data (17, 21). In a quantitative comparison of mRNA levels of brain and inner medulla (Fig. 2B), we found that inner medulla has approximately one-third alpha 1G mRNA relative to that of brain. Comparisons of alpha 1G mRNA levels were made by using beta -actin-normalized values.


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Fig. 2.   alpha 1G mRNA level in the rat kidney IM compared with various rat tissues. A: autoradiograph of an RNase protection assay for alpha 1G mRNA in various tissues: heart (left ventricle), brain (cerebral cortex), lung, skeletal muscle, and kidney IM. B: autoradiograph of an RNase protection assay for alpha 1G mRNA in brain and IM. C: beta -actin-normalized values of protected alpha 1G mRNA in brain and IM.

Nephron distribution of alpha 1G mRNA. To determine in which segment(s) along the rat nephron alpha 1G is expressed, we used the dual approach of RT-PCR and immunohistochemistry. RNA was extracted from microdissected nephron segments. By RT-PCR/Southern analysis, alpha 1G mRNA transcripts were consistently and abundantly detected in DCT, CT+CCD, and IMCD. Significant levels were observed in TL, mTALH, cTALH, and PST. Product was absent or at the limit of detection in all other segments tested. Rat beta -actin was equally amplified in all samples, with cDNA equivalent to 0.2-mm tubule length (Fig. 3A).


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Fig. 3.   Nephron distribution of alpha 1G mRNA in the rat kidney. A: ethidium bromide-stained agarose gels showing amplification products for alpha 1G and beta -actin (top and bottom, respectively) after RT-PCR (30 cycles) on microdissected nephron segments (1 and 0.2 mm of tubule length, respectively). Middle: autoradiograph of Southern analysis of RT-PCR using probe specific for alpha 1G. PCT, proximal convoluted tubule; PST, proximal straight tubule; DTL, descending thin limb of Henle's loop; TL, thin limb of Henle's loop from inner medulla; mTALH and cTALH, medullary and cortical thick ascending limb of Henle's loop, respectively; DCT, distal convoluted tubule; CT+CCD, connecting tubule and cortical collecting duct; OMCD and IMCD, outer medullary and inner medullary collecting duct, respectively. B: original gel showing amplified cDNA for the mouse alpha 1G-subunit. RNA obtained from whole kidney (positive control) and from the murine IMCD cell line mIMCD-3 was used as a template for RT-PCR (30 cycles). C: autoradiograph of an RNase protection assay for alpha 1G mRNA in mIMCD-3 cells.

PCR amplification of alpha 1G subunit cDNA from the IMCD-derived murine cell line mIMCD-3 resulted in abundant amplification product (Fig. 3B), suggesting that alpha 1G is expressed in the epithelial cells of the IMCD as opposed to adhering vascular or interstitial cells. The finding was further confirmed by RNase protection assay on mRNA harvested from the mIMCD-3 cells by using the rat riboprobe (Fig. 3C). In this latter assay, the intensity of full-length protected probe was quite faint, and bands of lower molecular sizes appeared. This is most likely due to incomplete hybridization of the rat probe to murine alpha 1G mRNA. Thus we did not attempt to quantify the expression of alpha 1G mRNA in the mIMCD-3 cells.

Localization of alpha 1G-subunit in rat kidney with immunoperoxidase microscopy and laser confocal microscopy. For immunohistochemistry, antibodies recognizing the NH2-terminal domain of the alpha 1G-subunit were raised. As demonstrated in Figs. 4 and 5, immunohistochemistry revealed that significant labeling was associated with DCT, CT, and IMCD in rat kidney. In contrast, glomeruli, proximal tubules, thick ascending limbs, and CCD and OMCD were unlabeled. In the rat kidney cortex, distinct labeling was associated with DCT and CT segments (Fig. 4, A-D). The labeling was restricted to the very apical plasma membrane domains (arrows in Fig. 4, A-D). Immunolabeling controls using peptide-absorbed antibodies revealed no labeling in rat kidney cortex (Fig. 4E). In kidney inner medulla, prominent labeling was associated with IMCD, where the labeling was also confined to the apical plasma membrane domains of principal cells (arrows, Fig. 5A). This distribution pattern was confirmed by immunohistochemistry using anti-aquaporin-2, which labeled principal cells in parallel semithin cryosections (not shown). Labeling was also observed in thin limbs (Fig. 5A). Immunolabeling control using peptide-absorbed antibodies revealed no labeling in inner medulla (Fig. 5B).


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Fig. 4.   Immunohistochemical analyses of the cellular and subcellular localization of the alpha 1G-subunit in rat kidney cortex using immunoperoxidase labeling of semithin paraffin sections. A-C: in rat renal cortex, alpha 1G-subunit labeling was associated with apical plasma membrane domains of the CT (arrows). D: in cortex, alpha 1G-subunit labeling was also associated with apical plasma membrane domains of the DCT (arrows). No significant labeling was observed in proximal tubules or in glomeruli (Glom). E: section from rat kidney cortex. Control using peptide-absorbed antibodies revealed no immunolabeling.



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Fig. 5.   Immunohistochemical analyses of the cellular and subcellular localization of the alpha 1G-subunit in rat kidney IM by using immunoperoxidase labeling of semithin paraffin sections. A: in kidney IM, strong labeling was associated with apical plasma membrane domains of IMCD principal cells (arrows). Labeling was also observed in thin limbs (arrow, bottom right). B: section from rat kidney IM. Control using peptide-absorbed antibodies revealed no immunolabeling.

As a control to the localization in kidney cortex, an additional series of experiments was done. Serial sections were labeled with anti-alpha 1G and anti-calbindin D-28K (Fig. 6, A and B). Calbindin is expressed in DCT cells and principal cells of the CT, where it is involved in controlled calcium reabsorption. It was confirmed that alpha 1G is coexpressed with calbindin in CT (Fig. 6, A and B), which allows the conclusion that alpha 1G is at least expressed in principal cells of the CT. Labeling with anti-H+-ATPase was also performed (Fig. 6, C-F). The H+-ATPase beta 1-subunit is expressed in the intercalated cells of the late DCT, CT, and collecting duct. Anti-H+-ATPase antibody labeled scattered cells in DCT and CT segments, in accordance with labeling of the intercalated cells (Fig. 6, D and F). The anti-alpha 1G antibody labeled apical membrane domains in DCT and CT cells, sparing only a few cells (Fig. 6, C and E). In serial sections, the H+-ATPase-positive cells were different from alpha 1G-positive cells (Fig. 6, C-F). Thus immunolabeling of serial sections of rat kidney cortex strongly supports the view that alpha 1G is expressed in the apical membrane domains of DCT cells and principal cells of the CT, whereas no labeling of alpha 1G was observed at the intercalated cells in these segments. Moreover, double-labeling laser confocal microscopy of alpha 1G with vacuolar H+-ATPase further demonstrated that, in DCT and CT, alpha 1G is apically expressed in principal cells with no labeling of intercalated cells (double-labeled for H+-ATPase in the same section, data not shown). Thus the alpha 1G-subunit is exclusively expressed in the apical membrane domains of DCT cells and principal cells of the CT as well as of the IMCD.


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Fig. 6.   Immunohistochemical analyses of the cellular localization of the alpha 1G-subunit compared with calbindin D28K and the beta 1-subunit of the H+-ATPase in rat kidney cortex using immunoperoxidase labeling of semithin serial paraffin sections. In rat renal cortex, alpha 1G-subunit labeling was associated with apical plasma membrane domains in the majority of cells of the connecting tubules (* and arrows in A and E) and DCT cells (C). Calbindin D-28K labeling (* in B) colocalizes with alpha 1G in the CT (* and arrows in A) as shown in serial sections (A and B). H+-ATPase labeling was observed in a few scattered intercalated cells of the DCT (* , arrows, and arrowheads in D) and CT (arrows in F). In serial sections (C-F), alpha 1G was observed in the DCT cells and principal cells of the CT (arrows in C and E), whereas H+-ATPase was only observed in the intercalated cells in these segments (arrows and arrowheads in D and F).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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Recently, a novel alpha 1-subunit of the voltage-gated calcium channel family was cloned (21). The alpha 1G-subunit shows T-type characteristics in the expression systems tested so far; i.e., it has a low single-channel conductance and is activated at membrane potentials of -60 mV. Northern blotting has shown that alpha 1G mRNA is expressed in rat, human, and mouse kidney (17, 21). The purpose of the present study was to determine the regional and nephron distribution pattern of alpha 1G in the rat kidney. The results confirmed that alpha 1G is expressed in the rat kidney and, in addition, showed a high level of heterogeneity for alpha 1G along the cortical-medullary axis. Expression of alpha 1G mRNA was strongest in the inner medulla. RT-PCR analysis of microdissected nephron segments showed that alpha 1G mRNA localized to the DCT, CT and CCD, and the IMCD, whereas the OMCD was negative. The localization of the alpha 1G mRNA to the IMCD epithelial cells was confirmed by detection in a mouse IMCD cell line.

Immunostaining with an antibody raised against a synthetic peptide confirmed the presence of alpha 1G protein in DCT, CT, and IMCD and extended the observation to demonstrate that the labeling was localized to the apical membrane domain of the cells. There was no apparent immunostaining for alpha 1G in the PST, cTAL, and CCD segments, although these cortical structures did express alpha 1G mRNA as determined by RT-PCR. This apparent discrepancy most likely reflects the different sensitivities of the methods used for detection. The specificity of the reaction was confirmed by preincubation of the antibody with the peptide used for immunization. Immunostaining of the brain (not shown) exactly reproduced the published localization of alpha 1G protein (7).

Most of the epithelial cells in the apical membranes of DCT and CT were labeled with the alpha 1G antibody, which colocalized with calbindin D28K immunoreactivity (Figs. 4 and 6, A and B). This allows the conclusion that the DCT cells and principal cells of CT express the alpha 1G-subunit protein. Identification of intercalated cells by an antibody against the H+-ATPase showed scattered positive foci primarily in the CT segment. There was no obvious colocalization with alpha 1G in serial sections (Fig. 6, C-F), and, in addition, the outer medullary collecting duct, which is rich in intercalated cells, was completely unlabeled with the alpha 1G antibody. It should be noted that these results were obtained with separate, but adjacent, serial sections. True colocalization with double immunofluorescence labeling essentially confirmed these data. Thus the alpha 1G-subunit is exclusively expressed in the apical membrane domains of DCT cells and principal cells of the CT as well as of the IMCD.

The only known signal for activation of T-type channels is depolarization of the cells. Because the T-type channels are already activated at -60 mV, and because they rapidly inactivate, they are not likely to play any functional role in nephron segments where the apical membrane potential is more positive than this value. In late CT and cortical collecting duct, the membrane potential is more positive because of a dominant role of the electrogenic sodium channel (ENaC) in epithelial transport. In contrast, DCT, early CT, and IMCD cells have rather negative apical membrane potential (5, 28) because both the NaCl cotransporter and ENaC are involved in NaCl absorption. On the basis of these considerations, depolarization could theoretically activate apical T-type channels in DCT, CT, and IMCD. These segments are exactly the sites where we observed expression of alpha 1G-subunits. In these segments depolarization could be mediated by an increase in sodium absorption through ENaC, by basolateral chloride efflux in DCT (23) and IMCD cells (4, 14), or by ischemic injury in the inner medulla, leading to cell swelling (5) and calcium influx through voltage-dependent calcium channels (20). Other signals may also be relevant. The extracellular calcium receptor (CaR) is expressed in IMCD (25) and in DCT and CT (29). Several mechanisms are likely to be involved in the rise in intracellular calcium, after activation of the CaR, and it is interesting to note that activation of voltage-dependent calcium channels has been suggested in various cell types. (8, 19).

Hormonally regulated calcium absorption is accomplished probably mainly by calcium entry via apical endothelial calcium (ECaC) channels of DCT and CT (12, 13). The ECaC is dihydropyridine insensitive. However, a number of reports exist of calcium uptake in DCT cells, which are sensitive to dihydropyridines (2, 9, 10, 18), in concentrations where they may also affect T-type channels (1, 27). Thus the presence of T-type channels in these membrane segments may provide an explanation for some of the data on apical calcium channels in DCT cells that are not explained by the ECaC channel.

In conclusion, we have provided evidence for the expression of a low-voltage-activated calcium channel alpha 1G-subunit in the apical membranes of DCT, CT, and IMCD cells.


    ACKNOWLEDGEMENTS

The authors thank Mette Fredenslund, Inger-Merete Paulsen, Zhila Nikrozi, and Mette Vistisen for expert technical assistance, and Anthony M. Carter for critical reading. We thank Dr. Henrik Hager, Dept. of Cell Biology, Univ. of Aarhus, for help on the laser scanning confocal microscopy.


    FOOTNOTES

Support for this study was provided by the Danish Health Science Research Council (9802855, 9601829,9503037), Ruth E. König Petersens Foundation, Novo Nordisk Foundation, Danish Heart Foundation (98-1-2-22583), Knud Øster-Jørgensens Foundation, Karen Elise Jensen Foundation, University of Southern Denmark Research Foundation, University of Aarhus Research Foundation, the University of Southern Denmark, the University of Aarhus, and the EU-Commission (EU-TMR and EU-Biotech programs).

Address for reprint requests and other correspondence: B. L. Jensen, Dept. of Physiology and Pharmacology, Winsløwparken 21.3, DK-5000 Odense C, Denmark (E-mail: bljensen{at}health.sdu.dk).

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.

Received 8 December 1999; accepted in final form 17 August 2000.


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Am J Physiol Renal Fluid Electrolyte Physiol 279(6):F997-F1005
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