ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain.

KEY POINTS: Gain-of-function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM. ABSTRACT: Skeletal muscle differentiation relies on store-operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1-/- /Stim2-/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters.

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

Zinc transporters in prostate cancer.

Prostate cancer is a major health concern as it has the second highest incidence rate among cancers in men. Despite progress in tumor diagnostics and therapeutic approaches, prognosis for men with advanced disease remains poor. In this review we provide insight into the changes of the intermediary metabolism in normal prostate and prostate cancer. In contrast to normal cells, prostate cancer cells are reprogrammed for optimal energy-efficiency with a functional Krebs cycle and minimal apoptosis rates. A key element in this relationship is the uniquely high zinc level of normal prostate epithelial cells. Zinc is transported by the SLC30 and SLC39 families of zinc transporters. However, in prostate cancer the intracellular zinc content is remarkably reduced and expression levels of certain zinc transporters are altered. Here, we summarize the role of different zinc transporters in the development of prostate cancer.

A two-part model for reference curve estimation subject to a limit of detection.

Reference curves are commonly used to identify individuals with extreme values of clinically relevant variables or stages of progression which depend naturally on age or maturation. Estimation of reference curves can be complicated by a technical limit of detection (LOD) that censors the measurement from the left, as is the case in our study of reproductive hormone levels in boys around the time of the onset of puberty. We discuss issues with common approaches to the LOD problem in the context of our pubertal hormone study, and propose a two-part model that addresses these issues. One part of the proposed model specifies the probability of a measurement exceeding the LOD as a function of age. The other part of the model specifies the conditional distribution of a measurement given that it exceeds the LOD, again as a function of age. Information from the two parts can be combined to estimate the identifiable portion (i.e. above the LOD) of a reference curve and to calculate the relative standing of a given measurement above the LOD. Unlike some common approaches to LOD problems, the two-part model is free of untestable assumptions involving unobservable quantities, flexible for modeling the observable data, and easy to implement with existing software. The method is illustrated with hormone data from the Third National Health and Nutrition Examination Survey.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis.

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Inherited epithelial transporter disorders--an overview.

In the late 1990s, the identification of transporters and transporter-associated genes progressed substantially due to the development of new cloning approaches such as expression cloning and, subsequently, to the implementation of the human genome project. Since then, the role of many transporter genes in human diseases has been elucidated. In this overview, we focus on inherited disorders of epithelial transporters. In particular, we review genetic defects of the genes encoding glucose transporters (SLC2 and SLC5 families) and amino acid transporters (SLC1, SLC3, SLC6 and SLC7 families).

[Physiology and biochemistry of uric acid]. / Physiologie und Biochemie der Harnsäure.

In humans, uric acid is the final breakdown product of unwanted purine nucleotides. Uric acid is the last stage in purine degradation, because humans lack the enzyme uricase which converts uric acid into allantoin. Uric acid has profound beneficial effects since it scavenges potential harmful radicals in our body. However, in conjunction with genetic or environmental factors, uric acid can cause significant health problems, leading to kidney stones when it builds up in the kidneys and to gout when crystals accumulate in the joints. The levels of uric acid in the blood must be tightly controlled to minimize these detrimental effects. Normally, the body eliminates enough uric acid in the kidney, and in part also through the intestines, to keep its concentration at a healthy level in the blood (approximately 300 microM). In patients with gout or kidney stone disease, however, the body either produces excessive amounts of uric acid or its ability to eliminate uric acid is disturbed in some way. In the kidney, uric acid is reabsorbed via the uric acid transporter URAT1. This transporter is the major mechanism for regulating blood uric acid levels and therefore may prove an interesting target for future drug development.

Modulation of DMT1 activity by redox compounds.

Iron(II) exacerbates the effects of oxidative stress via the Fenton reaction. A number of human diseases are associated with iron accumulation including ischemia-reperfusion injury, inflammation and certain neurodegenerative diseases. The functional properties and localization in plasma membrane of cells and endosomes suggest an important role for the divalent metal transporter DMT1 (also known as DCT1 and Nramp2) in iron transport and cellular iron homeostasis. Although iron metabolism is strictly controlled and the activity of DMT1 is central in controlling iron homeostasis, no regulatory mechanisms for DMT1 have been so far identified. Our studies show that the activity of DMT1 is modulated by compounds that affect its redox status. We also show that both iron and zinc are transported by DMT1 when expressed in Xenopus laevis oocytes. Radiotracer uptake and electrophysiological measurements revealed that H(2)O(2) and Hg(2+) treatments result in substantial inhibition of DMT1. These findings may have a profound relevance from a physiological and pathophysiological standpoint.

Effect of breastfeeding on cognitive development of infants born small for gestational age.

UNLABELLED: Breastfeeding during infancy appears to result in enhanced cognitive development during childhood, but it is not known whether breastfeeding should be encouraged for infants born small for gestational age (SGA) whose growth might otherwise benefit from nutritional supplementation. To address this issue, duration of exclusive breastfeeding and cognitive development were evaluated prospectively for 220 term children born SGA and 299 term children born appropriate for gestational age (AGA). Cognitive development was assessed using the Bayley Scale of Infant Development at 13 mo and Wechsler Preschool and Primary Scales of Intelligence at 5 y of age. Infants born SGA were given supplemental foods significantly earlier than those born AGA. Growth of infants born SGA was not related to early nutritional supplementation. The salutary effect of exclusive breastfeeding on cognitive development was greater for children born SGA than for those born AGA. Based on a linear association between duration of exclusive breastfeeding and intelligence quotient (IQ), children born SGA and exclusively breastfed for 24 wk were predicted to have an 11-point IQ advantage over those breastfed for 12 wk, as opposed to a 3-point advantage for children born AGA with similar durations of breastfeeding. The IQ distribution of children born SGA and exclusively breastfed for more than 12 wk was not different from that of all children born AGA. CONCLUSION: Duration of exclusive breastfeeding has a significant impact on cognitive development without compromising growth among children born SGA. These data suggest that mothers should breastfeed exclusively for 24 wk to enhance cognitive development.

Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Single-channel activities of the human epithelial Ca2+ transport proteins CaT1 and CaT2.

The human epithelial channels, CaT1 and CaT2, were expressed in oocytes, and their single-channel characteristics were compared. In the presence of Na+ and K+ as charge carriers in the pipette solutions, channel activities were observed only when the the extracellular sides of the patches were exposed to nominally Ca2+- and Mg2+-free solutions. In patches of both CaT1- and CaT2-expressing oocytes, multiple channel openings were observed, but the current levels were higher in CaT2-expressing oocytes, particularly at more negative voltages. With K+ as a charge carrier in patches of CaT1-expressing oocytes, the channel activity was low at -10 to -60 mV, but increased dramatically at more negative potentials. This voltage dependence was observed in the presence of both Na+ and K+. The channel activity with Na+, however, was higher at all potentials. Differences between the voltage dependencies for the two cations were also observed in CaT2-expressing oocytes, but the channel activities were higher than those in CaT1-expressing oocytes, particularly in the presence of Na+. We also found that low concentrations of extracellular Mg2+ (5-50 microm) elicited a strong inhibitory action on the CaT channels. Activation of the CaT1 and CaT2 channels by hyperpolarization and other factors may promote increased Ca2+ entry that participates in stimulation of intestinal absorption and renal reabsorption and/or other Ca2+ transport mechanisms in epithelial cells.

Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters.

Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.

Are maternal and sonographic factors associated with the detection of a fetal echogenic cardiac focus?

OBJECTIVE: To evaluate the influence of maternal weight and the orientation of the fetal 4-chamber heart view on the detection of a fetal echogenic cardiac focus. METHODS: In this nested case-control study, 103 women undergoing anatomic surveys at 15 to 22 weeks between January 1, 1997, and June 15, 1999, were identified as having an echogenic cardiac focus via our computerized database. A control group was selected from among the same group of patients. Data were collected from the sonography reports, prenatal records, and sonographic images of 4-chamber heart views; maternal characteristics and sonographic details were recorded, including the orientation of the 4-chamber view (apical, basilar, and right and left lateral). RESULTS: Gravidas in the echogenic cardiac focus group were more likely to be of lower weight (68.0 +/- 14.4 versus 72.9 +/- 18.3 kg; P = .04), of lower body mass index (25.5 +/- 5.3 versus 27.3 +/- 6.2 kg/m2; P = .03), of younger age (24.4 +/- 6.5 versus 26.9 +/- 6.9 years; P = .01), and African American or Asian (37.9% versus 27.2% and 9.7% versus 2%; P = .01). Cases were scanned at earlier gestational ages (18.9 +/- 1.6 versus 19.5 +/- 1.7 weeks; P = .01). The focus group was more likely to have had an apical view of the fetal heart taken (80.8% versus 51.4%; P = .0001). Controls were more likely to have had a right lateral view taken (44.6% versus 20.8%; P = .002). No significant difference was found between groups in terms of any other maternal or sonographic variable studied. CONCLUSIONS: The echogenic cardiac focus group was more likely to have a lower body mass index and to be scanned with the apical fetal heart view. The orientation of the fetal 4-chamber heart view exerted the most statistically significant influence on detection rates for the echogenic cardiac focus, implying that the more technically facile the sonographic study, the more likely an echogenic cardiac focus will be found.

Structural conservation of the genes encoding CaT1, CaT2, and related cation channels.

We report here the genomic structures of the genes encoding human calcium transport proteins CaT1 and CaT2, which belong to a recently identified class of highly selective calcium entry channels. The mRNA for CaT1 was expressed more abundantly than that for CaT2 in three major tissues involved in transcellular calcium transport, namely intestine, kidney, and placenta, as determined by quantitative PCR. The genes encoding CaT1 and CaT2, ECAC2 and ECAC1, respectively, are completely conserved in terms of exon size in the coding regions. They also share similar intron-exon structures with the genes encoding the closely related, nonselective cation channels VR1, VRL-1, OTRPC4 (also known as VR-OAC, Trp12, and VRL-2), and a hypothetical protein, VRL-3. We conclude that ECAC2 and ECAC1, which encode calcium selective channels, share a common ancestral gene with the genes encoding the related nonselective cation channels.

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax-6 binding sites, which can drive eye-specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum [Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999) Nature 402: 370-371]. We obtained seven independent integration events among 41 fertile G0 Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac-mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector-marker system for effective gene transfer in a broad range of insect species.

CaT1 expression correlates with tumor grade in prostate cancer.

Ca(2+) signaling is important for growth and survival of prostatic carcinoma (PCa) cells. Here we report that the gene for CaT1, a channel protein highly selective for Ca(2+), is expressed at high levels in human PCa and in the LNCaP PCa cell line. CaT1 mRNA levels were elevated in PCa specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlated with Gleason grade in a PCa series. CaT1 mRNA was suppressed by androgen and was induced by a specific androgen receptor antagonist in LNCaP cells, suggesting that the gene is negatively regulated by androgen. These findings are the first to implicate a Ca(2+) channel in PCa progression and suggest that CaT1 may be a novel target for therapy.

Association between infant breastfeeding and overweight in young children.

CONTEXT: It has been suggested that breastfeeding is protective against children becoming overweight, and that there is a dose-dependent effect of its duration. OBJECTIVE: To determine whether breastfeeding and its duration are associated with a reduced risk of being overweight among young children in the United States. DESIGN AND SETTING: Data on infant feeding and child overweight status were taken from the third National Health and Nutrition Examination Survey (NHANES III), a cross-sectional health examination survey conducted from 1988-1994. SUBJECTS: Sample of 2685 US-born children between the ages of 3 and 5 years, with birth certificates, height and weight measures, and information on infant feeding. MAIN OUTCOME MEASURES: A body mass index (BMI) between the 85th and 94th percentile was considered at risk of overweight and a BMI in the 95th percentile or higher was considered being overweight. RESULTS: After adjusting for potential confounders, there was a reduced risk of being at risk of overweight for ever breastfed children (adjusted odds ratio [AOR], 0.63; 95% confidence interval [CI], 0.41-0.96) compared with those never breastfed. There was no reduced risk of being overweight (AOR, 0.84; 95% CI, 0.62-1.13). There was no clear dose-dependent effect of the duration of full breastfeeding on being at risk of overweight or overweight and no threshold effect. The strongest predictor of child overweight status was the mother's concurrent weight. The rate of children being overweight nearly tripled with maternal overweight status (BMI, 25.0-29.9 kg/m(2); AOR, 2.95; 95% CI, 1.35-6.42) and more than quadrupled with maternal obesity status (BMI >/=30.0 kg/m(2); AOR, 4.34; 95% CI, 2.50-7.54). CONCLUSIONS: There are inconsistent associations among breastfeeding, its duration, and the risk of being overweight in young children. Breastfeeding continues to be strongly recommended, but may not be as effective as moderating familial factors, such as dietary habits and physical activity, in preventing children from becoming overweight.

Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences expression of MHC class 1 molecules.

BACKGROUND & AIMS: hPepT1 is an intestinal epithelial apical membrane transporter responsible for uptake of di/tripeptides (including bacterial derived proinflammatory n-formyl peptides). hPepT1 expression normally has a strict axial gradient-highest in the proximal small intestine with no expression in the colon. METHODS: Small intestinal-like cells (Caco2-BBE), and colonic-like cells (HT29-Cl.19A), and colonic mucosa from diseased and control patients were used in the present study. RESULTS: hPepT1 expression occurs aberrantly in the colon with chronic ulcerative colitis (6 patients) and Crohn's disease (4 patients), but not in normal colon (4 patients) or colon with microscopic colitis (4 patients). To model expression of hPepT1 by colonic-like cells in inflamed states, we stably transfected HT29-Cl.19A cells with a modified hPepT1 tagged on the N-terminus with green fluorescence protein. Analysis of transfected cells revealed that: GFP-hPepT1 protein, like the natural protein, is targeted to the apical plasma membrane. In addition, the tagged protein retains the capability of di/tripeptide absorption, and the expression of the tagged protein by HT29-Cl.19A cells permits absorption of N-formyl-methionyl-leucyl-phenylalanine (fMLP), as occurs in hPepT1 expressing Caco2-BBE cells. fMLP uptake by colonic cells expressing GFP-hPepT1 specifically enhances major histocompatibility complex class I surface expression. CONCLUSIONS: These data collectively indicate that, in some states of chronic inflammation, hPepT1 may be anomolously expressed in the colon. Further, transport of fMLP by hPepT1 potentially stimulates expression of key accessory immune molecule, MHC-1.

Differential distribution of the glutamate transporters GLT-1 and GLAST in tanycytes of the third ventricle.

The ventral one-third of the ventricular lining in the hypothalamus is formed by specialized ependymal cells called the tanycytes. These cells may serve a neuroendocrine transport function because of their structural specializations, which include apical microvili on the ventricular surface and long basal processes that terminate on blood vessels or on the glia limitans. Here, we describe the expression of mRNA and protein for the glutamate transporters GLT-1 and GLAST in unique tanycyte populations of the third ventricle in rat brain. Using nonisotopic in situ hybridization, we demonstrate GLAST mRNA labeling in tanycytes of the ventral floor and lateral walls in the tuberal and mammillary recess portions of the third ventricle. This GLAST mRNA labeling had a higher intensity than the labeling intensity observed in regular ependymal cells throughout the ventricular system. Furthermore, we have identified strong GLT-1 mRNA labeling in a population of tanycytes situated in the dorsolateral walls of caudal tuberal and mammillary recess portions. Immunocytochemical staining indicates that both GLT-1 and GLAST protein are expressed in the tanycyte populations as well. These data corroborate previous findings that third ventricle tanycytes are functionally heterogeneous. Furthermore, the GLT-1-expressing tanycytes represent a population of tanycytes that, to date, has not been recognized as functionally distinct. The strong GLAST expression by the ventral tanycytes in the hypophysiotropic area suggests a role of tanycyte-mediated glutamate transport in neuroendocrine activity. The functional role of GLT-1 in dorsal wall tanycytes remains to be explored.

CaT1 manifests the pore properties of the calcium-release-activated calcium channel.

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.