Epithelial Ca Research Articles

Specific expression and regulation of glucose transporters in zebrafish ionocytes

Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na(+),K(+)-ATPase-rich (NaR), Na(+)-Cl(-) cotransporter-expressing (NCC), H(+)-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca(2+) uptake, Cl(-) uptake, Na(+) uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca(2+) or low Cl(-) freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca(2+) channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na(+), a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Expression regulation of Na+-K+-ATPase α1-subunit subtypes in zebrafish gill ionocytes

In zebrafish (Danio rerio), six distinct Na+-K+-ATPase (NKA) alpha1-subunit genes have been identified, and four of them, zatp1a1a.1, zatp1a1a.2, zatp1a1a.4, and zatp1a1a.5, are expressed in embryonic skin where different types of ionocytes appear. The present study attempted to test a hypothesis of whether these NKA alpha1 paralogues are specifically expressed and function in respective ionocytes. Double fluorescence in situ hybridization analysis demonstrated the specific expression of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 in NKA-rich (NaR) cells, Na+-Cl- cotransporter (NCC)-expressing cells, and H+-ATPase-rich (HR) cells, respectively, based on the colocalization of the three NKA alpha1 genes with marker genes of the respective ionocytes (epithelial Ca2+ channel in NaR cells; NCC in NCC cells; and H+-ATPase and Na+/H+ exchanger 3b in HR cells). The mRNA expression (by real-time PCR) of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 were, respectively, upregulated by low-Ca2+, low-Cl-, and low-Na+ freshwater, which had previously been reported to stimulate uptake functions of Ca2+, Cl-, and Na+. However, zatp1a1a.4 was not colocalized with any of the three types of ionocytes, nor did its mRNA respond to the ambient ions examined. Taken together, zATP1a1a.1, zATP1a1a.2, and zATP1a1a.5 may provide driving force for Na+-coupled cotransporter activity specifically in NaR, NCC, and HR cells, respectively.

Regulation of renal outer medullary potassium channel and renal K(+) excretion by Klotho.

Klotho is an aging-suppression protein predominantly expressed in kidney, parathyroid glands, and choroids plexus of the brain. The extracellular domain of Klotho, a type-1 membrane protein, is secreted into urine and blood and may function as an endocrine or paracrine hormone. The functional role of Klotho in the kidney remains largely unknown. Recent studies reported that treatment by the extracellular domain of Klotho (KLe) increases cell-surface abundance of transient receptor potential vanilloid type isoform 5, an epithelial Ca(2+) channel critical for Ca(2+) reabsorption in the kidney. Whether Klotho regulates surface expression of other channels in the kidney is not known. Here, we report that KLe treatment increases the cell-membrane abundance of the renal K(+) channel renal outer medullary potassium channel 1 (ROMK1) by removing terminal sialic acids from N-glycan of the channel. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 at the extracellular surface prevents clathrin-mediated endocytosis of ROMK1 and leads to accumulation of functional channel on the plasma membrane. Intravenous administration of KLe increases the level of Klotho in urine and increases urinary excretion of K(+). These results suggest that Klotho may have a broader function in the regulation of ion transport in the kidney.

Nedd4‐2 and Nedd4 mediate degradation of TRPV6 and TRPV5

Nedd4 and Nedd4‐2 are archetypal HECT ubiquitin E3 ligases that dispose target proteins for degradation. Because of the proven roles of Nedd4‐2 in degradation of membrane proteins such as epithelial Na+ channel (ENaC), we examined the effect of Nedd4‐2 and Nedd4 on the epithelial Ca2+ channels TRPV6 and TRPV5, which are involved in transcellular Ca2+ transport in the intestine and kidney, respectively, using Xenopus laevis oocyte system. Co‐expression of TRPV6 with Nedd4‐2 or Nedd4 resulted in a 47.9±1.4% or 28.3±1.8% reduction in TRPV6‐mediated Ca2+ uptake. Similarly, a 51.9±1.7% or 26.2±4.8% decrease in TRPV5‐mediated Ca2+ uptake was observed when Nedd4‐2 or Nedd4 was co‐expressed. Corresponding decrease in TRPV6‐ or TRPV5‐mediated Na+ current by Nedd4‐2 was detected using voltage clamp technique. Western blot analysis revealed that TRPV6 and TRPV5 protein were decreased by Nedd4‐2 and Nedd4, respectively, in a dose‐dependent manner. The down‐regulation of TRPV6 by Nedd4‐2 appeared to be dependent on the ubiquitin ligase activity, because the C922S ubiquitin ligase deficient mutant of Nedd4‐2 failed to inhibit TRPV6. Furthermore, an increase in TRPV6‐mediated Ca2+ uptake was observed when the HECT domain of Nedd4‐2 was removed. The Ca2+ binding C2 domain of Nedd4‐2 was also dispensable in this regulation, because the Nedd4‐2 construct without C2 domain exhibited a stronger inhibitory effect on TRPV6 than the wild‐type Nedd4‐2. In conclusion, our data suggest that Nedd4‐2 and Nedd4 may be involved in the degradation of TRPV6 and TRPV5.

Rapid effects of 17β-estradiol on TRPV5 epithelial Ca 2+ channels in rat renal cells

The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca 2+ entry step in transcellular Ca 2+ re-absorption in the kidney. The regulation of transmembrane Ca 2+ flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD 2) cells at mRNA and protein levels. We demonstrate that 17β-estradiol (E 2) is involved in the regulation of Ca 2+ influx in these cells via the epithelial Ca 2+ channels TRPV5. By combining whole-cell patch–clamp and Ca 2+-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20–50 nM E 2 rapidly (<5 min) induced a transient increase in inward whole-cell currents and intracellular Ca 2+ via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E 2 in kidney cells. Furthermore, the results suggest calcitropic effects of E 2. The results are discussed in relation to present concepts of non-genomic actions of E 2 in Ca 2+ homeostasis.

Activation of the Ca 2+-sensing receptor stimulates the activity of the epithelial Ca 2+ channel TRPV5

The extracellular Ca 2+-sensing receptor (CaR) is a key-player in plasma Ca 2+ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca 2+ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca 2+-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca 2+ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca 2+ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca 2+ influx via a PMA-insensitive PKC isoform pathway.

Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1

Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1

Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo

Stanniocalcin (STC) formerly called hypocalcin or teleocalcin, is a 50-kDa disulfide-linked homodimeric glycoprotein that was originally identified in fish and secreted from the corpuscles of Stannius (CS). One of the main functions of STC-1 is Ca(2+) uptake inhibition; however, the mechanisms remain unknown. In the present study, we provide molecular evidence to elucidate how zebrafish STC-1 regulates Ca(2+) uptake in zebrafish embryos. In a wide variety of tissues including the kidney, brain, gill, muscle, and skin, zstc-1 was expressed. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater stimulated whole body Ca(2+) influx and zebrafish epithelial Ca(2+) channel (zECaC) mRNA expression, while downregulated zstc-1 expression. A morpholino microinjection approach was used to knockdown the zSTC-1 protein, and the results showed that the Ca(2+) content, Ca(2+) influx, and zECaC mRNA expression all increased in morphants. These data suggest that zSTC-1 negatively regulates ECaC gene expression to reduce Ca(2+) uptake in zebrafish embryos.

Cystic fibrosis and estrogens: a perfect storm

Irreversible destruction and widening of the airways due to acquired infections or genetic mutations as well as those of unknown cause are more severe in females. Differences between male and female anatomy, behavior, and hormonal state have been proposed to explain the increased incidence and severity in females with airway disease such as cystic fibrosis (CF); however, a mechanism to explain a sex-related difference has remained elusive. In this issue of the JCI, Coakley et al. report that elevations in the major estrogen hormone in humans--17beta-estradiol--reduce Ca2+-activated Cl- secretion by airway epithelial cells in culture, thereby disrupting ion and water balance (see the related article beginning on page 4025). They measure a similar diminution of nasal epithelial Ca2+-activated Cl- secretion in women with CF during the menstrual cycle phase at which 17beta-estradiol level is at its highest. These data suggest that for about one week of a four-week menstrual cycle, women with CF will have a reduced ability to efficiently clear airway secretions, the buildup of which is a hallmark of CF. The authors suggest that these data warrant the testing of antiestrogen therapy in females with CF and propose an alternative avenue for CF therapeutic development.

Active Ca2+ reabsorption in the connecting tubule

The kidney plays a crucial role in the maintenance of the body calcium (Ca(2+)) balance. Ca(2+) is an essential ion in all organisms and participates in a large variety of structural and functional processes. In mammals, active tubular Ca(2+) reabsorption is restricted to the distal part of the nephron, i.e., the late distal convoluted (DCT2) and the connecting tubules (CNT), where approximately 10-15% of the total Ca(2+) is reabsorbed. This active transcellular transport is hallmarked by the transient receptor potential vanilloid 5 (TRPV5) epithelial Ca(2+) channel, regulated by an array of events, and mediated by hormones, including 1,25-dihydroxyvitamin D(3), parathyroid hormone, and estrogen. Novel molecular mechanisms have been identified, such as the direct regulatory effects of klotho and tissue kallikrein on the abundance of TRPV5 at the apical membrane. The newly discovered mechanisms could provide potential pharmacological targets in the therapy of renal Ca(2+) wasting. This review discusses the three basic molecular steps of active Ca(2+) reabsorption in the DCT/CNT segments of the nephron, including apical entry, cytoplasmic transport, and basolateral extrusion of Ca(2+). In addition, an overview of the recently identified mechanisms governing this active Ca(2+) transport through the DCT2/CNT epithelial cells will be presented.

TRPV5: an ingeniously controlled calcium channel

Body Ca(2+) homeostasis is tightly controlled and slight disturbances in renal Ca(2+) reabsorption can lead to excessive urine Ca(2+) excretion and promote kidney stone formation. The epithelial Ca(2+) channel TRPV5 constitutes the rate-limiting step of active Ca(2+) reabsorption in the kidney. Elucidation of the molecular pathways controlling TRPV5 function provides important information for our understanding of renal Ca(2+) handling, since active Ca(2+) reabsorption fine-tunes the final amount of Ca(2+) excreted into the urine. Over the last years, the molecular regulation of TRPV5 has been dismantled in detail. Various calciotropic hormones, known to alter renal Ca(2+) reabsorption, affect the expression of TRPV5. Others stimulate the trafficking of TRPV5 to the plasma membrane, while a number of associated proteins and ions control channel activity at the plasma membrane. Dynamic cell surface presence of TRPV5 is largely mediated by endosomal recycling processes allowing internalized channels to reappear at the plasma membrane. We present recently identified factors shown to modulate TRPV5 activity by diverse mechanisms to ultimately control renal Ca(2+) handling. The selected factors include klotho, tissue kallikrein, pH, Ca(2+), Mg(2+), PIP(2) and WNK4. This review covers the distinctive properties and regulation of the highly Ca(2+)-selective TRPV5 channel and highlights the implications for our understanding of the process of Ca(2+) reabsorption.

Effects of hypothermia on gene expression in zebrafish gills:upregulation in differentiation and function of ionocytes as compensatory responses

Ectothermic vertebrates are different from mammals that are sensitive to hypothermia and have to maintain core temperature for survival. Why and how ectothermic animals survive, grow and reproduce in low temperature have been for a long time a scientifically challenging and important inquiry to biologists. We used a microarray to profile the gill transcriptome in zebrafish (Danio rerio) after exposure to low temperature. Adult zebrafish were acclimated to a low temperature of 12 degrees C for 1 day and up to 30 days, and the gill transcriptome was compared with that of control fish in 28 degrees C by oligonucleotide microarray hybridization. Results showed 11 and 22 transcripts were found to be upregulated, whereas 56 and 70 transcripts were downregulated by low-temperature treatment for 1 day and 30 days, respectively. The gill transcriptome profiles revealed that ionoregulation-related genes were highly upregulated in cold-acclimated zebrafish. This paved the way to investigate the role of ionoregulatory genes in zebrafish gills during cold acclimation. Cold acclimation caused upregulation of genes that are essential for ionocyte specification, differentiation, ionoregulation, acid-base balance and the number of cells expressing these genes increased. For instance, epithelial Ca2+ channel (EcaC; an ionoregulatory protein) mRNA increased in parallel with the level of Ca2+ influx, revealing a functional compensation after long-term acclimation to cold. Phosphohistone H3 and TUNEL staining showed that the cell turnover rate was retarded in cold-acclimated gills. Altogether, these results suggest that gills may sustain their functions by producing mature ionocytes from pre-existing undifferentiated progenitors in low-temperature environments.

WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12A family of cation-chloride cotransporters and the renal outer medullary K+ channel ROMK and Cl(-) channel SLC26A9. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 and TRPV5 were coexpressed in Xenopus laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 82.7 +/- 7.1% increase in TRPV5-mediated Ca2+ uptake was observed when WNK3 was coexpressed. A similar increase in TRPV5-mediated Na+ current was observed with the voltage-clamp technique. WNK3 also enhanced Ca2+ influx and Na+ current mediated by TRPV6, which is the closest homolog of TRPV5 that mediates active intestinal Ca2+ absorption. The kinase domain of WNK3 alone was sufficient to increase TRPV5-mediated Ca2+ transport, and the positive regulatory effect was abolished by the kinase-inactive D294A mutation in WNK3, indicating a kinase-dependent mechanism. The complexly glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3. The exocytosis of TRPV5 was increased by WNK3, and the effect of WNK3 on TRPV5 was abolished by the microtubule inhibitor colchicine. The increased plasma membrane expression of TRPV5 was likely due to the enhanced delivery of mature TRPV5 to the plasma membrane from its intracellular pool via the secretory pathway. These results indicate that WNK3 is a positive regulator of the transcellular Ca2+ transport pathway.

Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1

Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca(2+) channel TRPV5 by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of beta-galactoside alpha2,6-sialyltransferase (ST6Gal-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6Gal-1, but is restored by forced expression of recombinant ST6Gal-1. Thus, Klotho participates in specific removal of alpha2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho.

The -glucuronidase klotho exclusively activates the epithelial Ca2+ channels TRPV5 and TRPV6

Active Ca(2+) reabsorption in the kidney is facilitated by the epithelial transient receptor potential vanilloid Ca(2+) channel subtype 5 (TRPV5). The complex-glycosylated TRPV5 is expressed at the apical membrane of the renal distal convoluted tubule (DCT) cells where the pro-urine hormone klotho can stimulate its activity by N-oligosaccharide hydrolysis. This study investigates whether klotho and its closely related analogue, beta-glucuronidase, can activate other renal ion channels than TRPV5 expressed by DCT cells. To determine the specificity of this stimulatory effect of klotho and beta-glucuronidase, a selection of ion channels and transporters expressed in the kidney (TRPV4, TRPV5, TRPV6 and TRPM6) was screened in transfected HEK293 cells by using Ca(2+)-influx measurements. Klotho and beta-glucuronidase have been found to significantly increase the activity of TRPV5 and TRPV6, but had no effect on TRPV4 and TRPM6. Furthermore, deglycosylation by endoglycosidase-F also stimulated the activity of TRPV4, TRPV5 and TRPV6, but not of TRPM6. These results suggest a modulating effect for klotho primarily restricted to the epithelial Ca(2+) channels TRPV5 and TRPV6.

Enriched uranium affects the expression of vitamin D receptor and retinoid X receptor in rat kidney

An increasing awareness of the radiological impact of the nuclear power industry and other nuclear technologies is observed nowadays on general population. This led to renew interest to assess the health impact of the use of enriched uranium (EU). The aim of this work was to investigate in vivo the effects of a chronic exposure to EU on vitamin D 3 metabolism, a hormone essential in mineral and bone homeostasis. Rats were exposed to EU in their drinking water for 9 months at a concentration of 40 mg l −1 (1 mg/rat day). The contamination did not change vitamin D plasma level. Vitamin D receptor ( vdr) and retinoid X receptor alpha ( rxrα), encoding nuclear receptors involved in the biological activities of vitamin D, showed a lower expression in kidney, while their protein levels were paradoxically increased. Gene expression of vitamin D target genes, epithelial Ca 2+ channel 1 ( ecac1) and Calbindin-D28k ( cabp-d28k), involved in renal calcium transport were decreased. Among the vitamin D target organs examined, these molecular modifications occurred exclusively in the kidney, which confirms that this organ is highly sensitive to uranium exposure. In conclusion, this study showed that a chronic exposure to EU affects both mRNA and protein expressions of renal nuclear receptors involved in vitamin D metabolism, without any modification of the circulating vitamin D.

Identification of Nipsnap1 as a novel auxiliary protein inhibiting TRPV6 activity

The transient receptor potential vanilloid channels 5 and 6 (TRPV5/6) are the most Ca(2+)-selective channels within the TRP superfamily of ion channels. These epithelial Ca(2+) channels are regulated at different intra- and extracellular sites by the feedback response of Ca(2+) itself, calciotropic hormones, and by TRPV5/6-associated proteins. In the present study, bioinformatics was used to search for novel TRPV5/6-associated genes. By including pull-down assays and functional analysis, Nipsnap1-a hitherto functionally uncharacterized globular protein-was identified as a novel factor involved in the regulation of TRPV6. Electrophysiological recordings revealed that Nipsnap1 abolishes TRPV6 currents. Subsequent biotinylation assays showed that TRPV6 plasma membrane expression did not change in the presence of Nipsnap1, suggesting that TRPV6 inhibition by Nipsnap1 is independently regulated from reduced cell surface channel expression. In addition, semi-quantitative reverse transcriptase PCR and immunohistochemical labeling of Nipsnap1 indicated that Nipsnap1 is expressed in mouse intestinal tissues-where TRPV6 is predominantly expressed-but that it does not co-localize with TRPV5 in the kidney. In conclusion, this study presents the first physiological function of Nipsnap1 as an associated protein inhibiting TRPV6 activity that possibly exerts its effect directly at the plasma membrane.

WNK3 enhances TRPV5‐mediated calcium transport via a kinase‐dependent pathway

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12 family of cation‐chloride cotransporters and the renal outer medullary K+ channel ROMK. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 with TRPV5 were co‐expressed in X. laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 80.6±11.4% increase in TRPV5‐mediated Ca2+ uptake was observed when WNK3 was co‐expressed. Similar increase in TRPV5‐mediated Na+ current was observed with voltage clamp technique. WNK3 also enhanced Ca2+ influx mediated by TRPV6, which is the closest homologue of TRPV5 that mediated active intestinal Ca2+ absorption. The positive regulatory effect was abolished by the kinase‐inactivating D294A mutation in WNK3, indicating a kinase‐dependent mechanism. The fully glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3, indicating that WNK3 increases TRPV5 expression at the cell surface. In the presence of brefeldin A, WNK3 slowed the declining of TRPV5‐mediated Ca2+ uptake over a period of 6 hours, suggesting that WNK3 stabilizes TRPV5 at the plasma membrane. In contrast, WNK3 exerted no significant effect on the delivery of TRPV5 to the plasma membrane from its intracellular pool. These results indicate that WNK3 is a positive regulator of transcellular Ca2+ transport pathway. Supported by NIH grant DK072154.

Mechanisms and Regulation of Epithelial Ca2+ Absorption in Health and Disease

Ca2+ is essential for numerous physiological functions in our bodies. Therefore, its homeostasis is finely maintained through the coordination of intestinal absorption, renal reabsorption, and bone resorption. The Ca2+-selective epithelial channels TRPV5 and TRPV6 have been identified, and their physiological roles have been revealed: TRPV5 is important in final renal Ca2+ reabsorption, and TRPV6 has a key role in intestinal Ca2+ absorption. The TRPV5 knockout mice exhibit renal leak hypercalciuria and accordingly upregulate their intestinal TRPV6 expression to compensate for their negative Ca2+ balance. In contrast, despite their severe negative Ca2+ balance, TRPV6-null mice do not display any compensatory mechanism, thus resulting in secondary hyperparathyroidism. These results indicate that the genes for TRPV5 and TRPV6 are differentially regulated in human diseases associated with disturbed Ca2+ balance such as hypercalciuria, osteoporosis, and vitamin D-resistant rickets.