Epithelial Calcium Channel Research Articles

Intestinal calcium absorption: Molecular vitamin D mediated mechanisms

Rickets and hyperparathyroidism caused by a defective Vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25-(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min after oral gavage in two strains of VDR-knock out (KO) mice (Leuven and Tokyo KO) and observed a threefold lower area under the curve in both KO-strains. Moreover, we evaluated the expression of intestinal candidate genes, belonging to a new class of calcium channels (TRPV), involved in transcellular calcium transport. The calcium transport protein ECaC2 was more abundantly expressed at mRNA level than ECaC1 in duodenum, but both were considerably reduced (ECaC2 > 90%, ECaC1 > 60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was only significantly decreased in the Tokyo KO, whereas PMCA(1b) expression was normal in both VDR-KOs. In Leuven wild type mice, a high calcium diet inhibited (> 90%), and 1,25(OH)(2)D(3) or low calcium diet induced (sixfold) duodenal ECaC2 expression and, to a lesser degree, ECaC1 and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas both ECaC mRNA expressions remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular ECaC2 or TRPV6) is strongly vitamin D dependent and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D dependent active calcium absorption.

Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice

It is known that cyclosporine A (CsA) treatment induces high bone-turnover osteopenia and hypercalciuria. It has been proposed that down-regulation of renal calbindin-D28k by CsA results in renal calcium wasting. We investigated the role of the kidney and bone in CsA-induced hypercalciuria in calbindin-D28k knockout (KO) and wild-type (WT) mice. Two sets of experiments were performed. In experiment 1, KO and WT mice were treated with CsA 20 mg/kg/day intraperitoneally (IP) for 7 days. In experiment 2, to eliminate the CsA effect on bone resorption, pamidronate (APD) 2.5 mg/kg IP was given every 4 days with the first dose given 4 days prior to the 7-day course of CsA. Serum levels of creatinine, calcium, and osteocalcin, as well as renal calcium excretion were measured to assess CsA's effects on calcium homeostasis. Effects of CsA on the expression of calbindin-D28k, and two calcium channels in the apical membrane of the distal tubule, epithelial calcium channel (ECaC) and alpha1G-subunit of a voltage-dependent Ca channel (alpha1G), in the kidney were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). KO mice had a threefold increase in renal calcium excretion when compared with WT mice at the baseline. This difference disappeared when calcium load was reduced by overnight fasting. After the CsA treatment, both WT and KO mice had a significant increase of renal calcium excretion (urine Ca/Cr ratio in WT, 0.11 +/- 0.01 to 1.29 +/- 0.17; in KO, 0.39 +/- 0.04 to 1.18 +/- 0.13; both P < 0.01). CsA treatment decreased renal calbindin-D28k mRNA by 61%, but did not affect the expression of ECaC and alpha1G. Baseline serum osteocalcin level of KO mice was significantly lower than that of WT mice. After CsA treatment, both groups had a 50% increase in the serum osteocalcin level, indicating increased bone turnover. When mice were treated with both CsA and APD, the increase in serum osteocalcin level was prevented, and renal calcium excretion was significantly lower than that in mice treated with CsA alone. However, there was still a significant increase in the urine Ca/Cr ratio in WT and KO mice compared with pretreatment levels (urine Ca/Cr in WT, 0.11 +/- 0.01 to 0.76 +/- 0.05, P < 0.01; in KO, 0.39 +/- 0.05 to 0.79 +/- 0.06; P < 0.01). Calbindin-D28k KO mice have diet-dependent hypercalciuria and a lower bone turnover rate. CsA treatment suppresses the expression of calbindin-D28k in mice, but has no effects on ECaC and alpha1G gene expression at the mRNA level. The pathogenesis of CsA-induced hypercalciuria involves both down-regulation of calbindin-D28k with subsequent impaired renal calcium reabsorption and CsA-induced high turnover bone disease. Additionally, our results suggest that mechanism(s) independent of calbindin-D28k within the kidney also may contribute to the CsA-induced calcium leak.

Opening of ligand-gated cation channel families by calpain inhibitors

The class of Ca 2+-permeable cation channels is composed of large families with six transmembrane segments including transient receptor potential, vanilloid receptor (VR), polycystin, epithelial calcium channels and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins made us believe that the blockade of calpain opens the silent channels. Using 1 μM of blockers in whole cellular patch pipette fill we measured currents of Chinese hamster ovary cells transfected by VR-like 1 and 2, polycystin-2, or a MLS-like new member (MLS3S). Significant conductance of every clone with a characteristic rectification by blockers was demonstrated. The permeability of Ca 2+ to them is similar to that reported. Western blot suggested that blockers did not affect the assembly of the protein but enabled its cleavage. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function.

Gene Structure and Regulation of the Murine Epithelial Calcium Channels ECaC1 and 2

The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)2D3, including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)2D3. Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.

Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA(1b)) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)(2)D(3) injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

The epithelial calcium channel, ECaC1: molecular details of a novel player in renal calcium handling.

The epithelial calcium channel, ECaC1: molecular details of a novel player in renal calcium handling.

Calcitriol controls the epithelial calcium channel in kidney.

The recently cloned epithelial Ca2+ channel (ECaC), which is expressed primarily in 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-responsive Ca2+ -transporting epithelia, is postulated to constitute the rate-limiting step in active Ca2+ reabsorption. In the present study, the effect of 1,25(OH)(2)D(3) was investigated on ECaC mRNA and protein levels in kidneys of rats that were raised on a vitamin D-depleting diet. This diet decreased the serum 1,25(OH)(2)D(3) concentration significantly, which was accompanied by a marked drop in serum Ca2+ level. Both 1,25(OH)(2)D(3) and Ca2+ levels were normalized within 48 h after 1,25(OH)(2)D(3) administration. In 1,25(OH)(2)D(3)-deficient rats, ECaC mRNA and protein levels of the kidney cortex were significantly decreased compared with the repleted animals, suggesting that 1,25(OH)(2)D(3) exerts its stimulatory effect on Ca2+ reabsorption via increased ECaC expression. In agreement with this observation, the elucidated human ECaC promoter contains several consensus vitamin D-responsive elements. ECaC was restricted to the apical membrane of the distal part of the distal convoluted and the connecting tubule. This conclusion was based on only minor overlap with the localization of the thiazide-sensitive NaCl co-transporter and complete co-localization with the 1,25(OH)(2)D(3)-dependent Ca2+ binding protein, calbindin-D(28K). In conclusion, ECaC, present in the distal part of the nephron, is an important target for 1,25(OH)(2)D(3)-mediated Ca2+ reabsorption.

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+of the epithelial Ca2+channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+measurements:1. Currents through ECaC were dramatically inhibited if Ca2+was the charge carrier. This inhibition was dependent on the extracellular Ca2+concentration and occurred also in cells buffered intracellularly with 10mM BAPTA.2. Application of 30mM [Ca2]einduced in non-Ca2] buffered HEK 293 cells at −80mV an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 ±15nM/s (n= 18 cells) and a peak value of 891 ± 106nM. The peak of the concomitant current with a density of 12.3 ± 2.6pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+transient, as expected if the Ca2+transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]iby dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10mM BAPTA, inhibited currents through ECaC carried by either Na+or Ca2+] ions. Half maximal inhibition of Ca2+currents in the absence of monovalent cations occurred at 67nM (n between 6 and 8), whereas Na+currents in the absence of Ca2+] and Mg2+were inhibited with an IC50of 89nM (n between 6 and 10). Currents through ECaC in the presence of 1mM Ca2+and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]iwith an IC50of 82nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i(IC50= 123nM, n between 7 and 18).4. The sensitivity of ECaC currents in inside-out patches for [Ca2]iwas slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169nM if Na+was the charge carrier (n between 4 and 11) and 228nM at 1mM [Ca2]e(n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+(whole-cell configuration) or removal of Ca2+from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 ± 34s (n= 15) in whole-cell mode and after 135 ± 23s (n= 17) in inside-out patches.6. We conclude that influx of Ca2+through ECaC and [Ca2]iinduce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca2+from an internal Ca2+binding site at ECaC.

Kinetics of activation of a PKC-regulated epithelial calcium channel

The kinetics of calcium entry through regulated calcium channels in cultured renal proximal tubule cells was studied with Fura-2 fluorescence ratio imaging in single cells. The calcium entry was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12-myristat-13-acetate (PMA), similar to that observed for activation by osmo-mechanical stress. OAG (2.5μM) or PMA (0.5μM) activated calcium entry is characterized by a significant latency between agonist application and the response, whereas the effect of osmo-mechanical stress was immediate. This pre-response latency was 260 ± 70s with OAG stimulation and 79.2 ± 17.3s with PMA stimulation. Once a cell responds, the intracellular calcium level reaches a peak value within seconds. The cell response to agonist is independent of the response of neighboring cells. The response kinetics resembles those of the calcium sparks in excitable cells, except the response is much slower. In all cases, the response appears to be an all-or-none event, that is characteristics of an elementary binary switch. It is suggested that the binary response and the lack of coordinated response of calcium entry in single cells results from limited availability of the calcium channels and/or PKC that activates the channel. The experimental data could be fit to a single binary response mathematical model assuming each response reflected an elementary event of a single channel opening or a co-ordinated opening of a cluster of several channels.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.

A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Epithelial calcium transporter expression in human duodenum.

Calcium absorption in intestine and kidney involves transport through the apical membrane, cytoplasm, and basolateral membrane of the epithelial cells. Apical membrane calcium influx channels have recently been described in rabbit (epithelial calcium channel, ECaC) and rat (calcium transport protein, CaT1). We amplified from human duodenum a 446-base partial cDNA probe (ECAC2) having a predicted amino acid similarity of 97% to rat CaT1. Duodenum, but not ileum, colon, or kidney, expressed a 3-kb transcript. A larger transcript was also found in placenta and pancreas, and a different, faint transcript was found in brain. In duodenal biopsies from 20 normal volunteers, expression varied considerably but was not significantly correlated with vitamin D metabolites. This signal correlated with calbindin-D(9k) (r = 0.48, P < 0.05) and more strongly with the plasma membrane calcium ATPase PMCA1 (r = 0.83, P < 0.001). These data show that although individual variations in calcium channel transcripts are not vitamin D dependent, expression of genes governing apical entry and basolateral extrusion are tightly linked. This may account for some of the unexplained variability in calcium absorption.

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line.

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line. In the current study, we have demonstrated that Caco-2 cells express low but detectable levels of CaT1 mRNA in the absence of 1,25-dihydroxyvitamin D treatment. CaT1 mRNA expression is rapidly up regulated (4-fold increase at 4 h and 10-fold at 24 h) by treatment with 1,25-dihydroxyvitamin D (10(-7) moles/L). Moreover, the increase in CaT1 mRNA expression preceded by several hours the vitamin D induction of calbindin D9K, a putative cytosolic calcium transport protein. These observations are the first to demonstrate regulation of CaT1 expression by vitamin D and are consistent with a new model of intestinal calcium absorption wherein vitamin D-mediated changes in brush border membrane CaT1 levels could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.

Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells.

We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.

Gene Structure and Chromosomal Mapping of Human Epithelial Calcium Channel

The epithelial Ca2+ channel, ECaC, represents the rate-limiting step of vitamin D3-regulated Ca2+ (re)absorption in kidney and intestine, and provides, therefore, a new candidate gene for Ca2+-related disorders. To supply the basis for direct mutation analysis, we report here the structure of the human ECaC gene (ECAC12).It consists of 16 exons spanning 25 kb with introns ranging from 98 to 8500 bp. The 5′-flanking region of ECAC1 contains four putative vitamin D3-responsive elements. At positions −92 and −13 transcription initiation sites were identified, but the former lacks the canonical TATA or CAAT boxes. ECAC1 was mapped to chromosome 7q35 by fluorescent in situ hybridization, reassigning a previous radiation hybrid mapping to 7q31.1-2. The gene of a recently identified rat intestine homologue of ECaC, named Ca2+ transporter 1, was found juxtaposed to the ECaC gene, indicating that both genes are the products of evolutionary local gene duplication.

Epithelial calcium channel: gate-keeper of active calcium reabsorption

The epithelial calcium channel present in the apical membrane of 1,25-dihydroxyvitamin D3-responsive nephron segments represents the first member of a new family of calcium channels. This review covers the distinctive properties of this highly calcium-selective channel and highlights the implications for our understanding of the process of calcium reabsorption.

The Epithelial Calcium Channel, ECaC, Is Activated by Hyperpolarization and Regulated by Cytosolic Calcium

The recently cloned epithelial Ca2+ channel, ECaC, which is expressed in the apical membrane of 1,25-dihydroxyvitamin D3-responsible epithelia, was characterized in Xenopus laevis oocytes by measuring the Ca2+-activated Cl− current which is a sensitive read-out of the Ca2+ influx. ECaC-expressing oocytes responded to a voltage ramp with a maximal inward current of −2.1 ± 0.3 μA at a holding potential of −99 ± 1 mV. The inward current decreased progressively at less negative potentials and at +50 mV a small Ca2+-induced outward current was observed. The Ca2+ influx-evoked current at a hyperpolarizing pulse to −100 mV displayed a fast activation followed by a rapid but partial inactivation. Loading of the oocytes with the Ca2+ chelator BAPTA delayed the activation and blocked the inactivation of ECaC. When a series of brief hyperpolarizing pulses were given a significant decline in the peak response and subsequent plateau phase was observed. In conclusion, the distinct electrophysiological features of ECaC are hyperpolarization-dependent activation, Ca2+-dependent regulation of channel conductance and desensitization during repetitive stimulation.