COMMUNICATION
Molecular Identification of the Apical Ca2+
Channel in 1,25-Dihydroxyvitamin D3-responsive
Epithelia*
Joost G. J.
Hoenderop
§,
Annemiete W. C. M.
van der Kemp
,
Anita
Hartog
,
Stan F. J.
van de
Graaf
§,
Carel H.
van Os
,
Peter H. G. M.
Willems§, and
René J. M.
Bindels
¶
From the Departments of
Cell Physiology and
§ Biochemistry, Institute of Cellular Signaling, University
of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
 |
ABSTRACT |
In mammals, the extracellular calcium
concentration is maintained within a narrow range despite large
variations in daily dietary input and body demand. The small intestine
and kidney constitute the influx pathways into the extracellular
Ca2+ pool and, therefore, play a primary role in
Ca2+ homeostasis. We identified an apical Ca2+
influx channel, which is expressed in proximal small intestine, the
distal part of the nephron and placenta. This novel epithelial Ca2+ channel (ECaC) of 730 amino acids contains six
putative membrane-spanning domains with an additional hydrophobic
stretch predicted to be the pore region. ECaC resembles the recently
cloned capsaicin receptor and the transient receptor potential-related
ion channels with respect to its predicted topology but shares less
than 30% sequence homology with these channels. In kidney, ECaC is
abundantly present in the apical membrane of Ca2+
transporting cells and colocalizes with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K. ECaC
expression in Xenopus oocytes confers Ca2+
influx with properties identical to those observed in distal renal
cells. Thus, ECaC has the expected properties for being the gatekeeper
of 1,25-dihydroxyvitamin D3-dependent active
transepithelial Ca2+ transport.
 |
INTRODUCTION |
Calcium is the most abundant cation in the human body, but less
than 1% is present in ionic form in the extracellular compartment (1).
The extracellular Ca2+ concentration is precisely
controlled by parathyroid hormone (PTH)1 and
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3). Daily dietary intake is less
than 1000 mg of which only 30% is absorbed in the intestinal tract.
This percentage is significantly enhanced during growth, pregnancy, and
lactation by increased levels of circulating
1,25-(OH)2D3. Although there is a continuous
turnover of bone mass, there is no net gain or loss of Ca2+
from bone in a young and healthy individual. This implicates that
healthy adults excrete maximally 300 mg Ca2+ in the urine
to balance intestinal Ca2+ uptake and that the remaining
filtered load of Ca2+ has to be reabsorbed by the kidney.
Recently, the mechanism by which extracellular Ca2+ is
sensed by the parathyroid gland was elucidated by cloning of the
Ca2+-sensing receptor (2), and mutations in this receptor
gene explained familial hypocalciuric hypercalcemia (3). The importance of 1,25-(OH)2D3 in Ca2+ homeostasis
of the body is reflected by mutations in the genes coding for
1
-hydroxylase (4), a renal enzyme controlling its synthesis, and the
1,25-(OH)2D3-receptor (5). Transepithelial Ca2+ transport is a three-step process consisting of
passive entry across the apical membrane, cytosolic diffusion
facilitated by 1,25-(OH)2D3-dependent
calcium-binding proteins (calbindins), and active extrusion across the
opposing basolateral membrane mediated by a high affinity
Ca2+-ATPase and Na+-Ca2+ exchanger
(6). Until now, the molecular mechanism responsible for
Ca2+ entry into small intestinal and renal cells, which
serve as the influx pathways into the extracellular Ca2+
pool, is still elusive (6).
 |
EXPERIMENTAL PROCEDURES |
Primary Cultures of Kidney Cells--
Rabbit connecting tubule
(CNT) and cortical collecting duct (CCD) cells were immunodissected
from New Zealand White rabbits (~0.5 kg) with monoclonal antibody
R2G9, set in primary culture on permeable filter supports (0.33 cm2, Costar), and grown to confluence for 5 days, as
described previously (7).
Expression Cloning and DNA Analysis--
Poly(A)+
RNA, which induced 45Ca2+ uptake in
Xenopus laevis oocytes, was isolated from primary cultures
of rabbit CNT and CCD cells and used to construct a directional
cDNA library using a SuperScriptTM cDNA synthesis
system (Life Technologies, Inc.). cDNA was ligated into the pSPORT1
vector, and ElectroMax DH10B cells were transformed using a Bio-Rad
Gene Pulser. cRNA synthesized in vitro from pools of
~30.000 independent bacterial clones from this cDNA library was
injected in oocytes. A pool expressing highest
45Ca2+ uptake rates was sequentially subdivided
and analyzed until a single clone (ECaC) was identified that was
double-stranded sequenced using an automatic sequencer (ABI Prism 310 Genetic Analyzer). The mean hydrophobicity index was computed according
to the algorithm of Kyte and Doolittle (8) with a window of 9 residues.
Homology searches were performed against the nonredundant
GenBankTM data base.
Radioactive Ion Uptake in Oocytes--
Collagenase-treated
X. laevis oocytes were injected with 20 ng of in
vitro synthesized cRNA transcribed from pooled bacterial clones or
2 ng of in vitro synthesized cRNA from ECaC cDNA.
Ca2+ and Na+ uptake was determined 3 days after
injection by incubating 10-15 oocytes in 500 µl of medium (in
mM: 90 NaCl, 0.1 CaCl2, 1 µCi·ml
1
45Ca2+ or 0.4 µCi·ml
1
22Na+, 5 HEPES-Tris, pH 7.4) for 2 h at
18 °C. In the expression cloning experiments this medium was
supplemented with 10 µM felodipine, 10 µM
methoxyverapamil, 1 mM MgCl2, and 1
mM BaCl2. Each oocyte was washed three times in
stop buffer (in mM: 90 NaCl, 1 MgCl2, 0.5 CaCl2, 1.5 LaCl3, 5 HEPES-Tris, pH 7.4, 4 °C), solubilized with 10% (w/v) SDS, dissolved in scintillation
fluid, and counted for radioactivity.
Northern Analysis--
Poly(A)+ RNA (2.5 µg/lane)
was separated on a 18% (v/v) formaldehyde-1% (w/v) agarose gel and
blotted onto a nitrocellulose filter (Amersham Pharmacia Biotech). The
ECaC insert was excised from pSPORT1 and labeled with 32P
using a T7 QuickPrime kit (Amersham Pharmacia Biotech). Hybridization was for 16 h at 65 °C in 250 mM
Na2HPO4/NaH2PO4, pH
7.2, 7% (w/v) SDS, 1 mM EDTA, and filters were washed in
40 mM
Na2HPO4/NaH2PO4, pH
7.2, 0.1% (w/v) SDS, 1 mM EDTA for 20 min at 65 °C.
Immunohistochemistry--
Rabbit kidney slices were fixed in 1%
(v/v) periodate-lysine-paraformaldehyde fixative for 2 h, washed
with 20% (w/v) sucrose in phosphate-buffered saline, and subsequently
frozen in liquid N2. Sections (7 µm) were blocked with
5% (w/v) blocking reagent (NEN Life Science Products) in
phosphate-buffered saline for 15 min. Sections were washed three times
with Tris-buffered saline (TBS; 150 mM NaCl, 100 mM Tris-HCl, pH 7.5) and incubated with affinity-purified
guinea pig antiserum raised against the ECaC C-tail (amino acids
580-706) and rabbit anti-calbindin-D28K antiserum (9) for
16 h at 4 °C. After thorough washing with TBS, the sections
were incubated with the corresponding fluorescein isothiocyanate- or
tetramethylrhodamine isothiocyanate-conjugated anti-immunoglobulin G
for 60 min. Subsequently, sections were washed with TBS, distilled water, and methanol and finally mounted in Mowiol (Hoechst). All controls, including sections treated with preimmune serum or with conjugated antibodies only, were devoid of any staining.
Transcellular Ca2+ Transport--
Confluent
monolayers of rabbit CNT and CCD cells were washed twice and
preincubated in medium (in mM: 140 NaCl, 2 KCl, 1
K2HPO4, 1 MgCl2, 5 glucose, 5 L-alanine, 0.005 indomethacine, 0.0001 bovine PTH-(1-34),
10 HEPES-Tris, pH 7.4) containing 0.1 mM and 1
mM CaCl2 in the apical and basolateral
compartment, respectively, for 15 min at 37 °C. Subsequently, the
apical fluid was replaced with medium containing 1 µCi·ml
1
45Ca2+, and transcellular Ca2+ transport
was determined following removal of a 20-µl sample from the
basolateral medium at 30 min. The basolateral-to-apical flux was
negligible under all experimental conditions.
 |
RESULTS AND DISCUSSION |
Here, we report the expression cloning, tissue distribution,
immunolocalization, and functional characterization of the apical Ca2+ influx channel, which is expressed solely in proximal
small intestine, the distal part of the nephron, and placenta. In
analogy to the recently cloned amiloride-sensitive and
aldosterone-dependent epithelial Na+ channel
(ENaC) (10), present in the apical membrane of sodium-transporting epithelia, this novel epithelial Ca2+ channel was named
ECaC. By screening for maximal 45Ca2+ influx
activity in oocytes a single 2.8-kilobase pair cDNA was isolated
from a directional cDNA library prepared from poly(A)+
RNA of rabbit distal tubular cells. The ECaC cDNA contains an open
reading frame of 2190 nucleotides that encodes a protein of 730 amino
acids with a predicted relative molecular mass of 83 kDa
(Mr 83,000) (Fig.
1A). Hydropathy analysis
suggests that ECaC contains three structural domains: a large
hydrophilic amino-terminal domain of 327 amino acids containing three
ankyrin binding repeats and several potential protein kinase C
phosphorylation sites, suggesting an intracellular location; a six
transmembrane-spanning domain with two potential N-linked
glycosylation sites and an additional hydrophobic stretch between
transmembrane segments 5 and 6 indicative of an ion pore region;
and a hydrophilic 151-amino acid carboxyl terminus containing
potential protein kinase A and C phosphorylation sites (Fig.
1B).

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Fig. 1.
Molecular structure of epithelial
Ca2+ channel (ECaC). A, predicted amino
acid sequence encoded by the ECaC cDNA. Open boxes
delineate ankyrin repeat domains, black boxes predicted
transmembrane domains, the gray box a possible pore region,
and filled and open diamonds putative protein
kinase A and C phosphorylation sites, respectively. B,
predicted membrane topology and domain structure of ECaC. Outer and
inner plasma membrane leaflets are indicated. C, alignment
of ECaC pore region with that of related sequences. Identical residues
are in black boxes, and conservative substitutions are in
gray boxes. The GenBankTM accession numbers of
the rabbit ECaC, rat capsaicin receptor, Drosophila TRP
protein, and Caenorhabditis elegans olfactory channel are
AJ133128, AF029310, P19334, and AF031408, respectively.
|
|
A protein data base search revealed only a significant homology of less
than 30% between ECaC and the recently cloned capsaicin receptor (VR1)
(11), the transient receptor potential (TRP)-related ion channels (12)
and olfactory channels (13). The capsaicin receptor is a nonselective
cation channel and functions as a transducer of painful thermal stimuli
(11). Members of the TRP family have been proposed to mediate the entry
of extracellular Ca2+ into cells in response to depletion
of intracellular Ca2+ stores (12). These proteins resemble
ECaC with respect to their predicted topological organization and the
presence of multiple NH2-terminal ankyrin repeats (14).
There was also striking amino acid sequence similarity between ECaC,
VR1, and TRP-related proteins within and adjacent to the sixth
transmembrane segment, including the predicted area that may contribute
to the ion permeation path (Fig. 1C) (15). Outside these
regions, however, ECaC shares only 20% sequence similarity with VR1
and TRP family members, suggesting a distant evolutionary relationship
among these channels.
High stringency Northern blot analysis of ECaC transcripts revealed
prominent bands of ~3 kb in small intestine, kidney, and placenta
(Fig. 2). We found that in the intestine
ECaC mRNA expression was highest in duodenum, decreased in jejunum,
and absent in ileum and colon. ECaC mRNA expression in kidney and
placenta was comparable with jejunum. In addition, ECaC transcripts in
lung, skeletal muscle, stomach, heart, liver, spleen, and brain were
undetectable. Most important is that expression of ECaC coincides with
that of calbindin-D9K in intestine and placenta and
calbindin-D28K in kidney (16, 17).

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Fig. 2.
ECaC expression in small intestine, kidney,
and placenta. High-stringency Northern blot analysis of RNA from a
range of tissues probed with 32P-labeled ECaC cDNA.
Lanes were loaded with 2.5 µg of poly(A)+ RNA from rabbit
duodenum, jejunum, ileum, kidney, distal colon, lung, skeletal muscle,
stomach, heart, spleen, liver, brain, and human placenta, respectively.
Molecular mass standards (in kilobases) are indicated on the
left.
|
|
Immunofluorescence staining revealed that in kidney ECaC is abundantly
present along the apical membrane in the majority of cells lining the
distal part of the nephron including distal convoluted tubule,
connecting tubule and cortical collecting duct, where it colocalizes
with calbindin-D28K (Fig. 3).
This part of the nephron is the site of PTH- and
1,25-(OH)2D3-regulated transcellular Ca2+ reabsorption (18).

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Fig. 3.
Immunohistochemistry of ECaC in rabbit
kidney. Kidney cortex sections were double stained with anti-ECaC
antiserum (A, C) and
anti-calbindin-D28K (B, D).
Localization of ECaC in distal convoluted tubules (A) and
cortical collecting ducts (C) is restricted to the apical
region of the cell and colocalizes with calbindin-D28K. The
cells in the collecting duct lacking ECaC were also
calbindin-D28K-negative and are, therefore, most likely
intercalated cells. Note the absence of immunopositive staining of ECaC
in the surrounding glomeruli, proximal convoluted tubules, and thick
ascending limb of Henle's loop. Bar denotes 10 µm.
|
|
By functional expression of ECaC in Xenopus oocytes we
observed that the 45Ca2+ uptake was linear for
at least 2 h (Fig. 4) and increased
with increasing extracellular Ca2+ concentrations between
0.01 and 2.0 mM with an apparent affinity for
Ca2+ of ~0.2 mM (Fig.
5C). This is well within the
range of physiologically relevant extracellular calcium concentrations.
As shown in Fig. 5A, trivalent and divalent cations
inhibited 45Ca2+ influx in the following rank
order of potency: La3+ > Cd2+ > Mn2+, while Ba2+, Mg2+, and
Sr2+ had no effect. It is striking that Ba2+
and Sr2+, which are highly permeant in voltage-gated
Ca2+ channels (19), do not interfere with ECaC. In
addition, the L-type Ca2+ channel antagonists
and depolarization with 50 mM KCl were without effect. VR1,
to which ECaC has the highest homology, shows a relative low
permeability to monovalent ions such as Na+ (11). In a
double labeling experiment, ECaC-injected oocytes did not exhibit a
significant Na+ influx (0.88 ± 0.05 and 0.95 ± 0.05 nmol·h
1·oocyte
1 for ECaC and
water-injected oocytes, respectively; n = 29 oocytes; p > 0.2), while displaying a markedly increased
Ca2+ influx. In humans metabolic acidosis induces
hypercalciuria (18, 20), and here we demonstrate that acidification of
the extracellular medium to pH 5.9 significantly inhibits
45Ca2+ influx (Fig. 5, A and
B). If extrapolatable to the in vivo situation this effect could well be the molecular explanation of acidosis-induced calciuresis. Taken together, these characteristics indicate that ECaC
is distinct from previously described Ca2+ channels.
Furthermore, the above described pharmacological and functional
properties of ECaC are identical to those of Ca2+ transport
across the monolayers (Fig. 5, C and D),
providing evidence that the protein is a major constituent of the
transcellular Ca2+ transport system in renal cells.
Together with the previous finding that the Ca2+ influx
rate at the apical membrane of renal distal cells is tightly coupled to
transepithelial Ca2+ flux over a wide range of transport
rates (21), this suggests that the apical Ca2+ influx is
the rate-limiting step in transcellular Ca2+ transport.
Moreover, this implicates that hormonal regulation of a single influx
pathway, i.e. ECaC, may control the rate of transcellular
Ca2+ transport.

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Fig. 4.
45Ca2+ uptake in oocytes
expressing ECaC as a function of time. Measurements were performed
at the indicated time points in water-injected ( ) and ECaC-injected
( ) oocytes. Values are means ± S.E. of three
experiments.
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Fig. 5.
Functional characterization of ECaC.
A and B, substrate specificity, pH dependence,
and voltage dependence of 45Ca2+ uptake in
oocytes expressing ECaC (A) and transcellular
45Ca2+ transport across rabbit kidney cells
(B). Cations were added at a final concentration of 0.5 mM, pH was lowered from 7.4 to 5.9, 50 mM NaCl
was replaced by 50 mM KCl to depolarize the membrane,
methoxyverapamil (m-verapamil) and felodipine were added at
10 µM. In transcellular Ca2+ transport
measurements across renal cells these additions were applied to the
apical compartment only. C and D, concentration
dependence of 45Ca2+ uptake in oocytes
expressing ECaC (C) and transcellular
45Ca2+ transport in rabbit kidney cells
(D). Measurements were performed at the indicated
Ca2+ concentrations in extracellular and apical medium of
oocytes and confluent monolayers, respectively. Water-injected oocytes
exhibited a Ca2+ uptake of less than 6% of the
ECaC-injected oocytes. Values are means ± S.E. of five
experiments. Statistical significance (*, p < 0.05)
was determined by analysis of variance.
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|
In humans, approximately 4% of the population suffers from idiopathic
hypercalciuria with the characteristics of autosomal dominant
transmission (22). In some of the affected individuals, hypercalciuria
is secondary to hyperabsorption of Ca2+ (22). Gain of
function mutations in ECaC or its dysregulation may well be the cause
of absorptive hypercalciuria. The present elucidation of ECaC allows to
study these possibilities with molecular genetic approaches.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the valuable advice
of Dr. Jonathan Lytton in constructing the kidney cDNA library, the
superb assistance of Judith Nelissen with the sequence analysis, and
thank Drs. Jan Joep de Pont, Bé Wieringa, and Nine Knoers for
critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Dutch
Organization of Scientific Research (NWO-ALW 805-09.042).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.
¶
To whom correspondence should be addressed: 162 Cell
Physiology, University of Nijmegen, P. O. Box 9101, NL-6500 HB
Nijmegen, The Netherlands. Tel.: 31-24-3614211; Fax: 31-24-3540525;
E-mail: reneb{at}sci.kun.nl.
 |
ABBREVIATIONS |
The abbreviations used are:
PTH, parathyroid
hormone;
1, 25-(OH)2D3, 1,25-dihydroxyvitamin
D3;
CNT, connecting tubule;
CCD, cortical collecting duct;
ECaC, epithelial Ca2+ channel;
TRP, transient receptor
potential.
 |
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