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

Dominik Müller, Joost G. J. Hoenderop, Carel H. van Os and René J. M. Bindels

Department of Cell Physiology, University Medical Centre Nijmegen, The Netherlands

Keywords: calcitriol; kidney; duodenum; ECaC2; CaT1; calcium absorption

Introduction

The recent identification of the epithelial Ca2+ channel, ECaC1, in the kidney represents a major step forward in our knowledge of renal Ca2+ handling. This membrane channel protein is the first member of a new family of Ca2+-selective cation channels. It consists of six transmembrane-spanning domains, including a pore-forming hydrophobic stretch between domains 5 and 6. ECaC1 constitutes the apical entry mechanism of active, transcellular Ca2+ reabsorption. In contrast to the paracellular route, this transcellular pathway enables the organism to actively control the net amount of Ca2+ reabsorption. In vivo studies indicate a specific regulation of ECaC1 by calcitriol. This novel Ca2+ channel is also expressed in other tissues like intestine, pancreas, brain, testis and prostate. The central role of ECaC1 in active Ca2+ reabsorption makes it a prime target for pharmacological manipulation, and several disorders related to Ca2+ homeostasis could benefit from such developments. This review highlights the identification, characteristics and clinical impact of the epithelial calcium channel ECaC1.

Calcium handling by the kidney

The kidney plays an important role in mammalian Ca2+ homeostasis [1]. To maintain constant supply of Ca2+ with a minimal energetic effort, the main fraction of the tubular Ca2+ load is reabsorbed via a paracellular, passive flux that is driven by the existing electrochemical gradient in the proximal tubule and the thick ascending limb of Henle's loop, accounting for reabsorption of about 80–90% of the filtered Ca2+ [2]. Despite the fact that paracellular tight junction proteins like claudin-16 exhibit a certain ion-selectivity and that mutations in these proteins can cause diseases in humans, this pathway is not specifically controlled [3]. The final amount of Ca2+ excreted in the urine (normally less than 0.1 mmol/kg body weight/24 h is tightly regulated by a transcellular pathway in more distal neplron segments (Figure 1Go) [4]. In contrast to the paracellular route, only this transcellular pathway allows the body to regulate Ca2+ reabsorption independent of the Na+ balance. Via this process of fine-tuning, the organism can respond immediately to dietary fluctuations of nutritional Ca2+, while it can also adapt to long-term changes of the body's demand which occur during growth, development and ageing [5]. It can be anticipated that disturbances of active Ca2+ reabsorption are accompanied by significant alterations of the overall Ca2+ homeostasis. Active Ca2+ reabsorption has been investigated for many years, but only the recent molecular identification of the apical entry step of Ca2+-reabsorbing cells has made it possible to study the molecular details of Ca2+ metabolism in health and disease [6].



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Fig. 1. Ca2+ reabsorption along the tubule. The remaining Ca2+ at different sites of the nephron is indicated in percentages. Transcellular Ca2+ transport only takes place in the distal nephron (pink) and is carried out as a 3-step-process. Following entry of Ca2+ through the epithelial Ca2+ channel, ECaC1, cytosolic Ca2+ is buffered by calbindin-D28k. At the basolateral membrane, Ca2+ is extruded via a Ca2+-ATPase (PMCA1b) and a sodium-calcium-exchanger (NCX1). l,25(OH)2D3 regulates this process by stimulating the expression of ECaC1 and calbindin-D28k.

 
Transcellular Ca2+ transport in the kidney involves a 3-step process. After apical entry via ECaC1, cytosolic Ca2+ is buffered by a Ca2+-binding protein (calbindin-D28k) [2]. Following facilitated diffusion to the basolateral site, Ca2+ is extruded into the blood compartment by a Na+-Ca2+ exchanger (NCX1) and a Ca2+-ATPase (PMCA1b). In contrast to NCX, PMCA isoforms are ubiquitously expressed in nephron segments and are more likely to be involved in cellular Ca2+ homeostasis than specifically involved in active Ca2+ transport [7,8]. In the kidney, transcellular transport of Ca2+ only takes place in the distal part of the nephron which, based on functional and anatomical features, can be divided in two segments of the distal convoluted tubule (DCT1 and DCT2) and the connecting tubule (CNT) [9]. The importance of the distal nephron in regulating Ca2+ homeostasis is substantiated by the known hypocalciuric action of thiazide diuretics as well as the hypocalciuria seen in patients with Gitelman's syndrome, which is caused by mutations in the thiazide-sensitive NaC1 co-transporter (NCC) [10]. The steroid hormone calcitriol (1,25 (OH)2D3) has been shown to be effective in the distal nephron. In proximal tubular cells 1{alpha}-hydroxylase guarantees the body's supply of calcitriol, but in the distal nephron this enzyme may produce calcitriol in an autocrine and paracrine fashion to supply local targets [11].

Molecular details of the epithelial calcium channel, ECaC1

ECaC1 was first cloned by our group from rabbit kidney cortex by an expression-cloning strategy [6]. Next, we identified the human orthologue of ECaC1 from kidney [12]. At the amino acid level, human ECaC1 displays a homology of about 80% with rabbit ECaC1. A database search demonstrated a distant relation of ECaC1 to the transient receptor potential (TPR)-related ion channels and to receptors for different sensorial stimuli (OSM-9, vanilloid receptors) [1315]. The homology with these channels is as low as about 30%, which suggests that ECaC1 is the first member of a new class of ion channels.

The nucleotides of the 2187 bp open reading frame of ECaC1 encode a protein of 729 amino acids with a predicted molecular weight of 83 kDa. The putative secondary structure consists of six transmembrane-spanning domains with a short hydrophobic stretch between transmembrane domain 5 and 6 (Figure 2AGo). Based on the known structural details of other six membrane-spanning channel proteins, it can be anticipated that four monomers constitute a functional tetrameric channel [16]. Comparing different species, it was found that several putative regulatory elements of ECaC1 are conserved. These sites include six putative protein kinase C (PKC) phosphorylation sites, three ankyrin repeats, as well as a classical PDZ domain. The latter domain is an important motif in protein-protein interaction and might play a regulatory role in ECaC1 trafficking to the plasma membrane [17]. The physiological impact of these regulatory sites remains to be addressed in functional investigations.



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Fig. 2. (A) Predicted topology of ECaC1 consisting of six transmembrane spanning domains with a short hydrophobic stretch between transmembrane domain 5 and 6. Cytosolic C- and N-terminal tails contain several potential regulatory sites for PKC phosphorylation (yellow), ankyrin repeats (grey) and a PDZ domain (blue). (B) Schematic drawing of the gene structure of ECaC1 including the promoter region, exon 1 (including the 5'-UTR and the start of the open reading frame) and intron 1. The VDRE consensus sites are boxed in blue. Activator protein 1 and 2 (AP-1, AP-2) and stimulatory protein 1 (SP-1) consensus sequences are indicated. The transcription initiation site is marked by an arrow (+1).

 
To provide the basis for direct mutation screening in Ca2+-related candidate disorders, we recently elucidated the genomic organization of human ECaC1 [18]. The gene spans 25 kb on chromosome 7q35. In contrast to our previous finding, a major revision of this chromosomal area by the Human Genome Project revealed that ECaC1 consists of 15 exons rather than of 16 exons [18]. Computer analysis indicated that the ECaC1 promoter does not contain a canonical TATA- or CAAT-box, but displays several other putative binding sites for transcriptional activators that can be typically found in promoter regions, like AP-1/AP-2 and SP-l sites. Additionally, the promoter region of ECaC1 contains four consensus sequences that could serve as functional vitamin D-responsive elements (VDRE) (Figure 2BGo). These sites are located within the first 900 bp before the transcription initiation site, matching well with the average distance of functional elements from the transcription start point so far described [19,20].

On the mRNA level, human ECaC1 is, in addition to kidney, also expressed in small intestine, pancreas, brain, testis, prostate, colon ascendens and colon transversum. Interestingly, in all ECaC1-positive tissues, it co-localizes with the expression of the cytosolic Ca2+ binding protein, calbindin-D [12]. Immunohistochemical analysis demonstrated the presence of this channel in the apical membrane of DCT2 and CNT of the kidney cortex as well as in the apical part of the brush-border membrane of duodenal and jejunal villi [2] (Figure 3Go). In these tissues, we could also demonstrate co-localization of ECaC1 with the associated proteins of transcellular Ca2+ transport, NCX1, PMCA and calbindin-D. Using antibodies against NCC, it became clear that there is only a small stretch of overlap between NCC and ECaC1. The localization of ECaC1 is restricted to the last distal part of the DCT (DCT2) and the connecting tubule. This finding suggests that the natriuretic and hypocalciuric effects of thiazides do not reside within a single nephron segment [22].



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Fig. 3. (A) Nomarski light microscopic image of rabbit kidney cortex. (B) Immunopositive fluorescence staining of ECaC1, which was predominantly found along the apical membrane of the depicted CNT.

 
Until now, only rabbit ECaC1 has been subjected to detailed electrophysiological characterization. Heterologous expression of ECaC1 in human embryonic kidney cells (HEK 293) and subsequent patch-clamp analysis showed that ECaC1 is a constitutively active Ca2+-selective channel with unique electrophysiological features including inward rectification and Ca2+ -dependent feedback inhibition [23]. The large currents seen at negative membrane potentials in HEK 293 cells suggest that ECaC1 allows a substantial Ca2+ flux under physiological conditions. The data further demonstrate that increased levels of cytosolic Ca2+ rapidly inactivate the Ca2+ flux through the channel, a mechanism that prevents cell damage from Ca2+ overload. The selectivity for Ca2+ over other divalent cations questions the results of clinical absorption studies in which Sr2+ is used as a Ca2+ surrogate [24]. We postulate that Sr2+ may provide a tool to study paracellular, but not transcellular Ca2+ transport. It is also important to note that ECaC1 is impermeable for Mg2+. Therefore, the clinical observation that increased urinary Mg2+ concentrations enhance urinary Ca2+ excretion does not find an explanation in simultaneous flux of both ions through ECaC1.

Interestingly, the open probability of ECaC1 is increased at high extracellular pH, whereas acidosis (pH<6.0) reduces its Ca2+ conductance. It has been shown in the past that primary cultures from rabbit cortical collecting systems exhibit transcellular Ca2+ transport that is inhibited by a decrease in luminal pH [25]. Additionally, ECaC1 activity studies in Xenopus laevis oocytes and HEK 293 cells showed an inhibition by low extracellular pH values [26]. This data underlines the fact that ECaC1 exhibits significant pH sensitivity (Figure 4Go).



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Fig. 4. (A) pH dependence of Ca2+ reabsorption in primary cultures of CNT cells. (B) Ca2+ currents in ECaC1-expressing HEK 293 cells as measured by patch-clamp analysis.

 
To address the in vivo regulation of ECaC1, rats were fed a vitamin D3 depleting diet and we compared the expression of ECaC1 in these animals with their littermates, which were placed on the same diet, but were repleted with injections of calcitriol [27]. As expected, plasma Ca2+ levels dropped in vitamin D deficient animals, but could be completely restored within 2 days after the i.p. administration of calcitriol. Expression of ECaC1 mRNA was increased in the repleted group and this difference was even more striking at the level of protein expression. These findings, together with the above-mentioned VDREs in the ECaC1 promoter region, suggest a calcitriol-dependent regulatory pathway for ECaC1 in the kidney (Figure 1Go).

ECaC1 homologues

Recently, a second member of the ECaC family was identified, initially called ‘calcium transporter 1’ (CaT1) [28]. Originally cloned from rat intestine, also the mouse and the human orthologues of this protein have now been identified. The hCaT1 orthologue displays a homology with hECaC1 of approximately 80% at the amino acid level [29]. Most impressive is the fact that the pore-forming region in ECaC1 and CaT1 is entirely conserved among different species, which points to a crucial role of this area in Ca2+ gating [30]. As demonstrated by Northern blot analysis, human CaT1 is expressed in oesophagus, stomach, duodenum, proximal jejunum, liver, placenta, pancreas and to a lesser extent in kidney [29]. Furthermore, it was claimed that a second, novel CaT1 homologue was expressed in rat kidney, which was called ‘CaT2’. However, a GenBankTM search revealed that ‘CaT2’ simply represents the rat orthologue of ECaC1, as published previously [31]. We have demonstrated by genomic cloning that ECaC1 and CaT1 are products of distinct genes, rather than being splice variants [18]. Both genes are juxtaposed on chromosome 7q35, which suggests an evolutionary gene duplication event. In general, gene duplication provides one of the major driving forces in evolution, since both genes will acquire different functions and expression patterns by randomly occurring mutations [32]. Taken together, the high sequence similarities, tissue distribution and function as Ca2+ channels rather than as transporters, indicates a close relationship between both proteins. In this line of reasoning, CaT1 represents the second member of the ECaC family and should, therefore, be referred to as ECaC2. Indeed, the HGMW-approved symbol of the corresponding human gene has been named ECaC2 [33].

Clinical implications

As mentioned above, ECaC1 and ECaC2 exhibit distinct features, placing them into a new family of Ca2+-selective channels. They represent the molecular basis of the apical Ca2+ entry step in transcellular Ca2+ transport occurring in kidney and intestine. Since only this pathway allows active regulation of the net amount of Ca2+ entering or leaving the body, the delineation of the molecular mechanisms involved in its regulation will provide new insight into disturbances of Ca2+ homeostasis. In addition, more detailed information could also facilitate the development of pharmaceutical strategies to influence Ca2+ (re)absorption. A variety of diseases could possibly benefit from such developments. In the following paragraph, we address a few candidate disorders, which are frequently encountered in daily clinical practice.

Secondary hyperparathyroidism (SHPT) is frequently observed in patients with end-stage renal disease. Although not entirely understood, one of the main contributing factors is a decreased activity of the renal enzyme, 1{alpha}-hydroxylase, converting 25(OH)D3 to its active metabolite calcitriol [34]. In order to overcome this situation, the administration of calcitriol has— besides the administration of Ca2+-based phosphate binders—gained its place in the therapeutic regimen of these patients. Calcitriol suppresses efficiently the transcriptional rate of PTH messenger in the parathyroid glands, but increases at the same time intestinal absorption of Ca2+ leading to hypercalcaemia with adverse effects.

Detailed knowledge of the molecular basis of calcitriol action, however, is still elusive. It has been demonstrated that the gene for intestinal cytosolic calbindin-D9k in rats displays a putative VDRE in its promoter region [35]. However, despite the fact that expression of rat calbindin-D9k is upregulated by calcitriol, it has recently been shown that this consensus site within the promoter region is not functional and that transcription must be controlled in a non-conventional way [36]. The same was reported for the human basolateral PMCA1 [37] whose expression has long been known to be influenced by the administration of calcitriol. However, a recent study failed to identify a functional VDRE within the first 1.5 kB of the 5'-flanking region of exon 1 [38]. Therefore, a direct genomic action of calcitriol has not been unequivocally demonstrated on the proteins involved in intestinal transcellular Ca2+ transport. So far, renal calbindin-D28k is the only protein involved in renal transcellular Ca2+ transport for which a functional VDRE in the promoter region was identified [20].

Recently, new analogues of calcitriol were introduced, which have been shown to suppress PTH secretion and exhibit significantly less calcaemic activity than the parent compound calcitriol [3941]. These analogues could display altered transcriptional activation of calcitriol-sensitive target genes, but their exact mechanism of action is not clearly understood at present.

The identification of ECaC1 provides a new target for the study of the effects of calcitriol and its analogues on renal and intestinal Ca2+ handling in more detail than was possible until now. The four putative VDREs in the promoter region of ECaC1 suggest strongly that calcitriol plays a major regulatory role [18]. Therefore, further investigation concerning regulation of ECaC1 and ECaC2 by calcitriol and its analogues will broaden our knowledge about the specific effects of these drugs on active Ca2+ transport.

Disturbed Ca2+ homeostasis in SHPT and many other diseases is associated with various signs frequently encountered in end-stage renal disease, as for example metabolic acidosis, which further maintain the vicious circle of Ca2+ wasting and increasing PTH levels [42,43]. In patients with already pre-existing SHPT, metabolic acidosis increases the risk of severe complications during the course of the disease. Evidence has been provided that acidosis favours Ca2+ mobilization from bone, but this only explains the renal Ca2+ wasting in part [44]. The pH-sensitivity of ECaC1 might shed new light on this matter. Lowering the pH reduces cellular Ca2+ influx via ECaC1. This pH sensitivity of ECaC1 ranges within the urinary pH values observed in metabolic acidosis and could contribute to inhibition of Ca2+ reabsorption in the distal nephron, leading to a further loss of Ca2+. It is mandatory to investigate to which extent the inhibition of ECaC1 contributes to Ca2+ wasting in acidosis and how new specific therapeutic strategies can be developed to counteract this inhibitory effect.

Finally, increased urinary Ca2+ excretion is correlated with a higher risk of developing kidney stones and nephrocalcinosis [45]. Although it appears that hypercalciuria is in many situations a secondary event, idiopathic forms are also known. In both forms reduction of urinary Ca2+excretion, for example, by using the Ca2+ retaining effect of thiazides, has been shown to reduce the risk of developing kidney stone disease. The side effects, however, limit their therapeutic use [46]. Again, a specific stimulatory pharmaceutical agent to enhance ECaC1 function might be able to increase Ca2+ reabsorption. Based on the data we have obtained so far, it is of interest to investigate whether defects in ECaC1 can even be the primary cause of inherited forms of increased renal Ca2+ loss. Alternatively, the latter forms could also be caused by increased intestinal activity of ECaC, leading via increased absorption of Ca2+ to surpassing the tubular threshold for Ca2+ reabsorption and thus to hypercalciuria.

Conclusion

The identification of the epithelial Ca2+ channel, ECaC1, provides new opportunities to study the process of active Ca2+ transport in the kidney and intestine. This discovery is, therefore, relevant for Ca2+-related human disorders, and could ultimately result in the development of pharmacological tools to influence channel function.

Acknowledgments

D.M. was supported by a grant from the Deutsche Forschungsgemeinschaft (MU 1497 2-1). J.G.J.H. is recipient of a long-term EMBO fellowship (ALTF-160-2000). The authors acknowledge Dr B. Nilius and co-workers of the Department of Physiology, KU Leuven for the ongoing collaboration on the electrophysiological characterization of ECaC.

Notes

Correspondence and offprint requests to: René J. M. Bindels, 162 Cell Physiology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Email: reneb{at}sci.kun.n Back

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