Molecular Identification of the Apical Ca2+Channel in 1,25-Dihydroxyvitamin D3-responsive Epithelia

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. 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. 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) 1The 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.1The 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. 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 (1van Os C.H. Biochim. Biophys. Acta. 1987; 906: 195-222Crossref PubMed Scopus (114) Google Scholar, 2Brown E.M. Gamba G. Riccardi D. Lombarbi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar), and mutations in this receptor gene explained familial hypocalciuric hypercalcemia (3Pollak M.R. Brown E.M. Chou Y.H. Hebert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1237-1239Abstract Full Text PDF PubMed Scopus (897) Google Scholar). The importance of 1,25-(OH)2D3 in Ca2+ homeostasis of the body is reflected by mutations in the genes coding for 1α-hydroxylase (4Kitanaka S. Takeyama K. Murayama A. Sato T. Okumura K. Nogami M. Hasegawa Y. Niimi H. Yanagisawa J. Tanaka T. Kato S. N. Engl. J. Med. 1998; 338: 653-661Crossref PubMed Scopus (289) Google Scholar), a renal enzyme controlling its synthesis, and the 1,25-(OH)2D3-receptor (5Hughes M.R. Malloy P.J. Kieback D.G. Kesterson R.A. Pike J.W. Feldman D. O'Malley B.W. Science. 1988; 242: 1702-1705Crossref PubMed Scopus (418) Google Scholar). 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 (6Friedman P.A. Gesek F. Physiol. Rev. 1995; 75: 429-471Crossref PubMed Scopus (196) Google Scholar). 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 (6Friedman P.A. Gesek F. Physiol. Rev. 1995; 75: 429-471Crossref PubMed Scopus (196) Google Scholar).RESULTS AND DISCUSSIONHere, 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) (10Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (822) Google Scholar), 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 (M r 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).A protein data base search revealed only a significant homology of less than 30% between ECaC and the recently cloned capsaicin receptor (VR1) (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar), the transient receptor potential (TRP)-related ion channels (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar) and olfactory channels (13Colbert H.A. Smith T.L. Bargmann C.I. J. Neurosci. 1997; 17: 8259-8269Crossref PubMed Google Scholar). The capsaicin receptor is a nonselective cation channel and functions as a transducer of painful thermal stimuli (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). 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 (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). These proteins resemble ECaC with respect to their predicted topological organization and the presence of multiple NH2-terminal ankyrin repeats (14Michaely P. Bennet V. Trends Cell Biol. 1992; 2: 127-129Abstract Full Text PDF PubMed Scopus (179) Google Scholar). 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) (15Hardie R.C. Minke B. Trends Neurosci. 1993; 61: 371-376Abstract Full Text PDF Scopus (233) Google Scholar). 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 of in small intestine, kidney, and (Fig. We that in the intestine ECaC expression was in in and in and ECaC expression in kidney and was with In ECaC transcripts in and is that expression of ECaC with that of in intestine and and in kidney Calcium and Cell Scholar, M. R. J. Physiol. Google expression in small intestine, kidney, and placenta. Northern blot analysis of from a range of with ECaC with of from rabbit kidney, distal and human mass revealed that in kidney ECaC is abundantly present the apical membrane in the of cells the distal part of the nephron including distal connecting and cortical collecting colocalizes with (Fig. This part of the nephron is the of and Ca2+ D. The B. of ECaC in rabbit kidney. with and of ECaC in distal and cortical collecting is to the apical of the and colocalizes with calbindin-D28K. The cells in the collecting ECaC also and therefore, most cells. the of of ECaC in the proximal and of functional expression of ECaC in Xenopus oocytes we observed that the 45Ca2+ uptake was for (Fig. and increased with extracellular Ca2+ between and with an affinity for Ca2+ of This is within the range of extracellular calcium in 5 and 45Ca2+ influx in the of and no is striking that which in Ca2+ channels S. H. J. Physiol. Scopus Google Scholar), with ECaC. In the Ca2+ channel and with VR1, to which ECaC has the a relative to as Na+ (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). In a oocytes a significant Na+ influx and for ECaC and a increased Ca2+ In D. The B. Scholar, Full Text PDF PubMed Scopus Google Scholar), and we that of the extracellular to significantly influx (Fig. A to the in this be the molecular of these that ECaC is from Ca2+ channels. the and functional properties of ECaC identical to those of Ca2+ transport across the (Fig. C and that the protein is a of the Ca2+ transport in renal cells. with the that the Ca2+ influx the apical membrane of renal distal cells is to transepithelial Ca2+ a range of transport G. F. R. Os C.H. Cell 1997; PubMed Scopus Google Scholar), this suggests that the apical Ca2+ influx is the in Ca2+ transport. this implicates that of a single influx ECaC, may the of Ca2+ uptake in oocytes ECaC as a of the in and of three characterization of ECaC. A and and of 45Ca2+ uptake in oocytes ECaC and transport across rabbit kidney cells a concentration of was from to was by to the membrane, and In Ca2+ transport across renal cells these to the apical compartment C and concentration of 45Ca2+ uptake in oocytes ECaC and transport in rabbit kidney cells the Ca2+ in extracellular and apical of oocytes and oocytes a Ca2+ uptake of less than of the of was by analysis of of the from with the of M.J. The B. Scholar). In of the is to of Ca2+ M.J. The B. Scholar). of mutations in ECaC or its may be the of The present of ECaC to these with molecular 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) 1The 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.1The 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. 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 (1van Os C.H. Biochim. Biophys. Acta. 1987; 906: 195-222Crossref PubMed Scopus (114) Google Scholar, 2Brown E.M. Gamba G. Riccardi D. Lombarbi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar), and mutations in this receptor gene explained familial hypocalciuric hypercalcemia (3Pollak M.R. Brown E.M. Chou Y.H. Hebert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1237-1239Abstract Full Text PDF PubMed Scopus (897) Google Scholar). The importance of 1,25-(OH)2D3 in Ca2+ homeostasis of the body is reflected by mutations in the genes coding for 1α-hydroxylase (4Kitanaka S. Takeyama K. Murayama A. Sato T. Okumura K. Nogami M. Hasegawa Y. Niimi H. Yanagisawa J. Tanaka T. Kato S. N. Engl. J. Med. 1998; 338: 653-661Crossref PubMed Scopus (289) Google Scholar), a renal enzyme controlling its synthesis, and the 1,25-(OH)2D3-receptor (5Hughes M.R. Malloy P.J. Kieback D.G. Kesterson R.A. Pike J.W. Feldman D. O'Malley B.W. Science. 1988; 242: 1702-1705Crossref PubMed Scopus (418) Google Scholar). 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 (6Friedman P.A. Gesek F. Physiol. Rev. 1995; 75: 429-471Crossref PubMed Scopus (196) Google Scholar). 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 (6Friedman P.A. Gesek F. Physiol. Rev. 1995; 75: 429-471Crossref PubMed Scopus (196) Google Scholar). AND DISCUSSIONHere, 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) (10Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (822) Google Scholar), 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 (M r 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).A protein data base search revealed only a significant homology of less than 30% between ECaC and the recently cloned capsaicin receptor (VR1) (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar), the transient receptor potential (TRP)-related ion channels (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar) and olfactory channels (13Colbert H.A. Smith T.L. Bargmann C.I. J. Neurosci. 1997; 17: 8259-8269Crossref PubMed Google Scholar). The capsaicin receptor is a nonselective cation channel and functions as a transducer of painful thermal stimuli (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). 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 (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). These proteins resemble ECaC with respect to their predicted topological organization and the presence of multiple NH2-terminal ankyrin repeats (14Michaely P. Bennet V. Trends Cell Biol. 1992; 2: 127-129Abstract Full Text PDF PubMed Scopus (179) Google Scholar). 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) (15Hardie R.C. Minke B. Trends Neurosci. 1993; 61: 371-376Abstract Full Text PDF Scopus (233) Google Scholar). 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 of in small intestine, kidney, and (Fig. We that in the intestine ECaC expression was in in and in and ECaC expression in kidney and was with In ECaC transcripts in and is that expression of ECaC with that of in intestine and and in kidney Calcium and Cell Scholar, M. R. J. Physiol. Google revealed that in kidney ECaC is abundantly present the apical membrane in the of cells the distal part of the nephron including distal connecting and cortical collecting colocalizes with (Fig. This part of the nephron is the of and Ca2+ D. The B. of ECaC in rabbit kidney. with and of ECaC in distal and cortical collecting is to the apical of the and colocalizes with calbindin-D28K. The cells in the collecting ECaC also and therefore, most cells. the of of ECaC in the proximal and of functional expression of ECaC in Xenopus oocytes we observed that the 45Ca2+ uptake was for (Fig. and increased with extracellular Ca2+ between and with an affinity for Ca2+ of This is within the range of extracellular calcium in 5 and 45Ca2+ influx in the of and no is striking that which in Ca2+ channels S. H. J. Physiol. Scopus Google Scholar), with ECaC. In the Ca2+ channel and with VR1, to which ECaC has the a relative to as Na+ (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). In a oocytes a significant Na+ influx and for ECaC and a increased Ca2+ In D. The B. Scholar, Full Text PDF PubMed Scopus Google Scholar), and we that of the extracellular to significantly influx (Fig. A to the in this be the molecular of these that ECaC is from Ca2+ channels. the and functional properties of ECaC identical to those of Ca2+ transport across the (Fig. C and that the protein is a of the Ca2+ transport in renal cells. with the that the Ca2+ influx the apical membrane of renal distal cells is to transepithelial Ca2+ a range of transport G. F. R. Os C.H. Cell 1997; PubMed Scopus Google Scholar), this suggests that the apical Ca2+ influx is the in Ca2+ transport. this implicates that of a single influx ECaC, may the of Ca2+ uptake in oocytes ECaC as a of the in and of three characterization of ECaC. A and and of 45Ca2+ uptake in oocytes ECaC and transport across rabbit kidney cells a concentration of was from to was by to the membrane, and In Ca2+ transport across renal cells these to the apical compartment C and concentration of 45Ca2+ uptake in oocytes ECaC and transport in rabbit kidney cells the Ca2+ in extracellular and apical of oocytes and oocytes a Ca2+ uptake of less than of the of was by analysis of of the from with the of M.J. The B. Scholar). In of the is to of Ca2+ M.J. The B. Scholar). of mutations in ECaC or its may be the of The present of ECaC to these with molecular 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) (10Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (822) Google Scholar), 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 (M r 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. A protein data base search revealed only a significant homology of less than 30% between ECaC and the recently cloned capsaicin receptor (VR1) (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar), the transient receptor potential (TRP)-related ion channels (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar) and olfactory channels (13Colbert H.A. Smith T.L. Bargmann C.I. J. Neurosci. 1997; 17: 8259-8269Crossref PubMed Google Scholar). The capsaicin receptor is a nonselective cation channel and functions as a transducer of painful thermal stimuli (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). 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 (12Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). These proteins resemble ECaC with respect to their predicted topological organization and the presence of multiple NH2-terminal ankyrin repeats (14Michaely P. Bennet V. Trends Cell Biol. 1992; 2: 127-129Abstract Full Text PDF PubMed Scopus (179) Google Scholar). 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) (15Hardie R.C. Minke B. Trends Neurosci. 1993; 61: 371-376Abstract Full Text PDF Scopus (233) Google Scholar). Outside these regions, however, ECaC shares only 20% sequence similarity with VR1 and TRP family members, suggesting a distant evolutionary relationship among these channels. stringency Northern blot analysis of ECaC transcripts revealed prominent of in small intestine, kidney, and (Fig. We that in the intestine ECaC expression was in in and in and ECaC expression in kidney and was with In ECaC transcripts in and is that expression of ECaC with that of in intestine and and in kidney Calcium and Cell Scholar, M. R. J. Physiol. Google Scholar). revealed that in kidney ECaC is abundantly present the apical membrane in the of cells the distal part of the nephron including distal connecting and cortical collecting colocalizes with (Fig. This part of the nephron is the of and Ca2+ D. The B. Scholar). By functional expression of ECaC in Xenopus oocytes we observed that the 45Ca2+ uptake was for (Fig. and increased with extracellular Ca2+ between and with an affinity for Ca2+ of This is within the range of extracellular calcium in 5 and 45Ca2+ influx in the of and no is striking that which in Ca2+ channels S. H. J. Physiol. Scopus Google Scholar), with ECaC. In the Ca2+ channel and with VR1, to which ECaC has the a relative to as Na+ (11Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar). In a oocytes a significant Na+ influx and for ECaC and a increased Ca2+ In D. The B. Scholar, Full Text PDF PubMed Scopus Google Scholar), and we that of the extracellular to significantly influx (Fig. A to the in this be the molecular of these that ECaC is from Ca2+ channels. the and functional properties of ECaC identical to those of Ca2+ transport across the (Fig. C and that the protein is a of the Ca2+ transport in renal cells. with the that the Ca2+ influx the apical membrane of renal distal cells is to transepithelial Ca2+ a range of transport G. F. R. Os C.H. Cell 1997; PubMed Scopus Google Scholar), this suggests that the apical Ca2+ influx is the in Ca2+ transport. this implicates that of a single influx ECaC, may the of Ca2+ transport. In of the from with the of M.J. The B. Scholar). In of the is to of Ca2+ M.J. The B. Scholar). of mutations in ECaC or its may be the of The present of ECaC to these with molecular We the of Lytton in the kidney cDNA the of with the sequence and and for reading of the