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Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit β3 (Cavβ3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor-controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3 -deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.

Independent of its function as a subunit of voltage-gated Ca 2+ channels, the Cavβ3 subunit desensitizes fibroblasts and pancreatic β-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca 2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavβ subunits exist, Cavβ1, Cavβ2, Cavβ3, and Cavβ4. To investigate whether the other Cavβ subunits, like Cavβ3, can desensitize cells to IP3 and thereby modulate Ca 2+ signaling, we expressed the cDNAs of Cavβ1, Cavβ2, Cavβ3, and Cavβ4 in COS-7 cells lacking endogenous Cavβ proteins. ATP stimulation of these cells results in the release of Ca 2+ from intracellular stores. This receptor-mediated Ca 2+ release is significantly decreased by Cavβ3 but not by Cavβ1, Cavβ2, and Cavβ4. Electrophysiological recordings of voltage-dependent Ca 2+ currents from fibroblasts show a small Ca 2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavβ2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca 2+ release is not significantly increased in Cavβ2-deficient fibroblasts, in contrast to Ca 2+ release in Cavβ3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavβ3 that is not shared by other Cavβ subunits., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca 2+ -selective and has been implicated in mediating transcellular Ca 2+ transport and thus maintaining the Ca 2+ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Voltage-gated Ca 2+ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP 3 ). Similarly, Cavβ3 could affect cytosolic calcium concentration ([Ca 2 + ]) in pancreatic β-cells. In this study, we deleted the Cavβ3-encoding gene Cacnb3 in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca 2+ signaling, glucose-induced insulin secretion (GIIS), Cav channel activity, and gene expression in wild-type cells in which Cavβ3 and the IP 3 receptor were coimmunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, proprotein convertase subtilisin/kexin type-1, and nitric oxide synthase-1 in Cavβ3-knockout cells. In the absence of Cavβ3, Cav currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP 3 -dependent Ca 2+ release and the frequency of Ca 2+ oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP 3 receptor in isolated β-cells, controls IP 3 -dependent Ca 2+ -signaling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner., (© 2021 by the American Diabetes Association.)

Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.

Background & Aims: Intestinal Ca 2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca 2+ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca 2+ absorption during this critical period., Methods: Expression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Ca v 1.3 calcium channels. Femurs were analyzed by using micro-computed tomography and histology., Results: Net TRPV6-mediated Ca 2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca 2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca 2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Ca v 1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Ca v 1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca 2+ absorption in the ileum, and increased expression of renal Ca 2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Ca v 1.3 expression., Conclusions: We have detailed novel pathways contributing to transcellular Ca 2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca 2+ balance early in life., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

PKD2 and PKD1 genes are mutated in human autosomal dominant polycystic kidney disease. PKD2 can form either a homomeric cation channel or a heteromeric complex with the PKD1 receptor, presumed to respond to ligand(s) and/or mechanical stimuli. Here, we identify a two-residue hydrophobic gate in PKD2L1, and a single-residue hydrophobic gate in PKD2. We find that a PKD2 gain-of-function gate mutant effectively rescues PKD2 knockdown-induced phenotypes in embryonic zebrafish. The structure of a PKD2 activating mutant F604P by cryo-electron microscopy reveals a π- to α-helix transition within the pore-lining helix S6 that leads to repositioning of the gate residue and channel activation. Overall the results identify hydrophobic gates and a gating mechanism of PKD2 and PKD2L1.

Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in Drosophila melanogaster Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, our data identify Flower as a key protein mediating granule endocytosis., (© 2018 Chang et al.)

Voltage-gated calcium channels (Cavs) are major Ca 2+ entry pathways in excitable cells. Their β subunits facilitate membrane trafficking of the channel's ion-conducting α1 pore and modulate its gating properties. We report that one β subunit, β3, reduces Ca 2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP 3 ) formation. This effect requires the SH3-HOOK domain of Cavβ3, includes physical β3/IP 3 receptor interaction, and prevails when agonist-induced IP 3 formation is bypassed by photolysis of caged IP 3 . In agreement with β3 acting as a brake on Ca 2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of β3. To mediate specific physiological responses and to prevent Ca 2+ toxicity, cytoplasmic Ca 2+ signals must be tightly controlled. The described function of β3, unrelated to its function as a Cav subunit, adds to this tight control., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21-Ser-42 to be functionally important. We then found that deletion mutant Δ1-36 (TRPP3 missing fragment Met-1-Arg-36) has a similar function as wild-type TRPP3, whereas Δ1-38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1-Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The auxiliary subunit α 2 δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α 2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α 2 δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca 2+ and Ba 2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Ca v 1.3 channels normally coassemble with α 2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca 2+ currents. Ca 2+ - and voltage-dependent K + (BK) currents and the expression of the pore-forming BKα protein were normal. Ca v 1.3 and Ca v β2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Ca v 1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Ca v 1.3 channels, the largely extracellularly localized α 2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca 2+ channels and postsynaptic AMPA receptors., Significance Statement: Inner hair cells possess calcium channels that are essential for transmitting sound information into synaptic transmitter release. Voltage-gated calcium channels can coassemble with auxiliary subunit α 2 δ isoforms 1-4. We found that hair cells of the mouse express the auxiliary subunit α 2 δ2, which is needed for normal hearing thresholds. Using a mouse model with a mutant, nonfunctional α 2 δ2 protein, we showed that the α 2 δ2 protein is necessary for normal calcium currents and exocytosis in inner hair cells. Unexpectedly, the α 2 δ2 protein is moreover required for the optimal spatial alignment of presynaptic calcium channels and postsynaptic glutamate receptor proteins across the synaptic cleft. This suggests that α 2 δ2 plays a novel role in organizing the synapse., (Copyright © 2016 the authors 0270-6474/16/3611024-13$15.00/0.)

Calcium ions (Ca(2+)) are important in numerous signal transduction processes, including the development and differentiation of T cells in the thymus. We report that thymocytes have multiple types of pore-forming α subunits and regulatory β subunits that constitute voltage-gated Ca(2+) (Cav) channels. In mice, T cell-specific deletion of the gene encoding the β2 regulatory subunit of Cav channels (Cacnb2) reduced the abundances of the channels Cav1.2 and Cav1.3 (both of which contain pore-forming α1 subunits) and impaired T cell development, which led to a substantial decrease in the numbers of thymocytes and peripheral T cells. Similar to the effect of Cacnb2 deficiency, pharmacological blockade of pore-forming Cav1α subunits reduced the sustained Ca(2+) influx in thymocytes upon stimulation of the T cell receptor, decreased the abundance of the transcription factor NFATc3, inhibited the proliferation of thymocytes in vitro, and led to lymphopenia in mice. Together, our data suggest that Cav1 channels are conduits for the sustained Ca(2+) influx that is required for the development of T cells., (Copyright © 2015, American Association for the Advancement of Science.)

Aims: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown., Methods and Results: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn(2+)-quench microfluorimetry, we identified a background Ca(2+) entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca(2+) concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca(2+)-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure., Conclusions: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca(2+) cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Voltage gated calcium channels (Cav) are composed of up to five proteins: The ion conducting pore subunit α1 and the auxiliary subunits α2, δ, β, and γ. Recent reports show that Cavα1 and Cavβ comprise the calcium channel core complex and that β, α2 δ and γ may serve additional roles that are independent of the Cavα1 subunit. This short review will summarize these emerging functions.

The transient receptor potential cation channel subfamily V member 6 (TRPV6) gene and cDNA were identified 15 years ago and exceptional observations on TrpV6 proteins and their function as a Ca(2+)-selective cation channel have been made since then. In this review we will summarize recent studies regarding the genetics, biochemical properties, and physiological functions of murine and human TrpV6 channel proteins. We will focus on TRPV6 gene polymorphisms, the start of TRPV6 translation at a non-AUG codon and the functions of TRPV6 in intestinal Ca(2+) uptake, sperm maturation, and male fertility.

The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4b > β4a > β4e). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions.

TRPV6 channels function as epithelial Ca(2+) entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca(2+) entry to prevent deleterious Ca(2+) overload.

TRPV6 is considered the primary protein responsible for transcellular Ca2+ absorption. In vitro studies demonstrate that a negatively charged amino acid (D) within the putative pore region of mouse TRPV6 (position 541) is critical for Ca2+ permeation of the channel. To elucidate the role of TRPV6 in transepithelial Ca2+ transport in vivo, we functionally analyzed a TRPV6D541A/D541A knockin mouse model. After weaning, mice were fed a regular (1% wt/wt) or Ca2+-deficient (0.02% wt/wt) diet and housed in metabolic cages. Blood was sampled for Ca2+ measurements, and the expression of Ca2+ transport proteins was analyzed in kidney and duodenum. Intestinal 45Ca2+ uptake was measured in vivo by an absorption assay. Challenging the mice with the Ca2+-deficient diet resulted in hypocalcemia in wild-type and TRPV6D541A/D541A mice. On a low-Ca2+ diet both mouse strains displayed increased expression of intestinal TRPV6, calbindin-D(9K), and renal TRPV5. TRPV6D541A/D541A mice showed significantly impaired intestinal Ca2+ uptake compared with wild-type mice, and duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice. On a normal diet, serum Ca2+ concentrations normalized in both mouse strains. Under these conditions, intestinal Ca2+ uptake was similar, and the expression levels of renal and intestinal Ca2+ transport proteins were not affected. We demonstrate that TRPV6D541A/D541A mice exhibit impaired transcellular Ca2+ absorption. Duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice, albeit insufficient to correct for the diminished Ca2+ absorption. Under normal conditions, when passive Ca2+ transport is predominant, no differences between wild-type and TRPV6D541A/D541A mice were observed. Our results demonstrate a specific role for TRPV6 in transepithelial Ca2+ absorption.

Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca(2+) absorption by the epididymal epithelium, resulting in abnormally high Ca(2+) concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6(D541A) proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6(-/-)) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6(D541A) pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6(-/-) males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca(2+) concentration that is mirrored by a lack of Ca(2+) uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca(2+) handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6(D541A) pore mutant in epididymal epithelial cells.

Bone is the major store for Ca(2+) in the body and plays an important role in Ca(2+) homeostasis. During bone formation and resorption Ca(2+) must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca(2+) transport machinery in these bone cells. In this study, we examined the epithelial Ca(2+) channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca(2+) transport genes, calbindin-D(9k) and/or -D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+) ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6(D541A/D541A) mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca(2+) transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice., (Copyright © 2011 Wiley Periodicals, Inc.)

TRPV6 [transient receptor potential vanilloid 6] is a calcium ion (Ca²+)-selective channel originally identified in the duodenal epithelium and in placenta; replacement of a negatively charged aspartate in the pore-forming region with an uncharged alanine (D541A) renders heterologously expressed TRPV6 channels nonfunctional. We found that male, but not female, mice homozygous for this mutation (Trpv6(D541A/D541A)) showed severely impaired fertility. The motility and fertilization capacity of sperm were markedly reduced, despite intact spermatogenesis. Trpv6 was expressed in epididymal epithelium where the protein was detected in the apical membrane, whereas it was not expressed in spermatozoa or the germinal epithelium. The Ca²+ concentration of the fluid in the cauda epididymis of Trpv6(D541A/D541A) males was 10 times higher than that of wild-type mice, which was accompanied by a seven- to eightfold decrease in Ca²+ absorption through the epididymal epithelium and was associated with reduced sperm viability. Thus, appropriate Ca²+ absorption and a consequent TRPV6-mediated decrease in the extracellular Ca²+ concentration toward the distal segments of the epididymal duct are essential for the acquisition of basic functions and the survival of spermatozoa.

Local Ca(2+) signaling occurring within nanometers of voltage-gated Ca(2+) (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockout-controlled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of pore-forming alpha1 and auxiliary beta subunits, are embedded into protein networks that may be assembled from a pool of approximately 200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca(2+) concentration, including G protein-coupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca(2+)-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca(2+) signaling by Cav2 channels in the brain.

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features., (Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The survival of T lymphocytes requires sustained, Ca(2+) influx-dependent gene expression. The molecular mechanism that governs sustained Ca(2+) influx in naive T lymphocytes is unknown. Here we report an essential role for the beta3 regulatory subunit of voltage-gated calcium (Ca(v)) channels in the maintenance of naive CD8(+) T cells. Deficiency in beta3 resulted in a profound survival defect of CD8(+) T cells. This defect correlated with depletion of the pore-forming subunit Ca(v)1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca(2+) entry in CD8(+) T cells. Ca(v)1.4 and beta3 associated with T cell signaling machinery and Ca(v)1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca(2+) entry is controlled by a Ca(v)1.4-beta3 channel complex in T cells.

Background: The transient receptor potential, subfamily V, member 6 (TRPV6) is a Ca(2+) selective cation channel. Several studies have shown that TRPV6 transcripts are expressed in locally advanced prostatic adenocarcinoma, metastatic and androgen-insensitive prostatic lesions but are undetectable in healthy prostate tissue and benign prostatic hyperplasia. Two allelic variants of the human trpv6 gene have been identified which are transcribed into two independent mRNAs, TRPV6a and TRPV6b. We now asked, whether the trpv6a allele is correlated with the onset of prostate cancer, with the Gleason score and the tumour stage., Methods: Genomic DNA of prostate cancer patients and control individuals was isolated from resections of prostatic adenocarcinomas and salivary fluid respectively. Genotyping of SNPs of the TRPV6 gene was performed by restriction length polymorphism or by sequencing analysis. RNA used for RT-PCR was isolated from prostate tissue. Data sets were analyzed by Chi-Square test., Results: We first characterized in detail the five polymorphisms present in the protein coding exons of the trpv6 gene and show that these polymorphisms are coupled and are underlying the TRPV6a and the TRPV6b variants. Next we analysed the frequencies of the two TRPV6 alleles using genomic DNA from saliva samples of 169 healthy individuals. The homozygous TRPV6b genotype predominated with 86%, whereas no homozygous TRPV6a carriers could be identified. The International HapMap Project identified a similar frequency for an Utah based population whereas in an African population the a-genotype prevailed. The incidence of prostate cancer is several times higher in African populations than in non-African and we then investigated the TRPV6a/b frequencies in 141 samples of prostatic adenocarcinoma. The TRPV6b allele was found in 87% of the samples without correlation with Gleason score and tumour stage., Conclusion: Our results show that the frequencies of trpv6 alleles in healthy control individuals and prostate cancer patients are not significantly different. Although expression of trpv6 transcripts correlates with aggressive potential of prostate cancer, the TRPV6 genotype does not correlate with the onset of prostate cancer, with the Gleason score and the tumour stage.

Voltage-activated Ca(2+) channels comprise complexes of a pore-forming Ca(V)alpha(1) and auxiliary subunits Ca(V)beta, Ca(V)alpha(2)delta and sometimes Ca(V)gamma. The intracellular Ca(V)beta subunit assists in trafficking and surface expression of the Ca(V)alpha(1) subunit and can modulate biophysical properties of the Ca(2+) channel. Four genes, Ca(V)beta1-4, exist which confer different properties to Ca(2+) currents through the various Ca(V)alpha(1) subunits. Ca(2+) currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L-type Ca(V)alpha(1) subunit Ca(V)1.3, but associated Ca(V)beta subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Ca(V)beta subunits including Ca(V)beta2, but clear assignment of the Ca(V)beta1-4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Ca(V)beta3 knockout (Ca(V)beta3(-/-)) and Ca(V)beta4 mutant mice (Ca(V)beta4(lh/lh)), which had normal hearing. Recording voltage-activated Ba(2+) currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba(2+) current properties compared with their wild-type controls. Neonatal Ca(V)beta4(lh/lh) IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Ca(V)beta4(wt), altogether indicating the presence of Ca(V)beta4 in IHCs. Ba(2+) currents of Ca(V)beta3(-/-) OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba(2+) current activation, and increased inactivation compared with Ca(V)beta3(wt), pointing to a role of Ca(V)beta3 for OHCs. These results indicate that neither Ca(V)beta3 nor Ca(V)beta4 are indispensable for hair cell Ca(2+) currents but contribute to the overall current properties.

Receptor-operated Ca(2+) entry (ROCE) and store-operated Ca(2+) entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca(2+) stores that activates Ca(2+) entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.

Recently ORAI and STIM proteins were identified as components of the CRAC channel and the endoplasmic reticulum Ca(2+) sensor, respectively. The ORAI proteins share a predicted structure that includes four transmembrane domains with intracellular N- and C-termini. They share structural similarity with proteins of the tetraspanin superfamily which includes the gamma subunits of voltage-activated Ca(2+) channels (CaVgamma), the transmembrane AMPA regulatory proteins (TARPs), the claudins and the tumor-associated membrane proteins (TMPs). The mouse genome contains four genes which encode the ORAI1, ORAI2 and ORAI3 proteins and two genes which encode the type I single-pass transmembrane STIM1 and STIM2 proteins. ORAI2 transcripts are present in primary cortical neurons and ORAI1, 2, 3 and STIM1, 2 expression is readily detectable in CD3+/CD4+-, CD3+/CD8+-, and CD19+-lymphocytes as well as in mast cells from mouse.

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.

The stimulation of membrane receptors coupled to the phopholipase C pathway leads to activation of the Ca(2+) release-activated Ca(2+) (CRAC) channels. Recent evidence indicates that ORAI1 is an essential pore subunit of CRAC channels. STIM1 is additionally required for CRAC channel activation. The present study focuses on the genomic organization, tissue expression pattern, and functional properties of the murine ORAI2. Additionally, we report the cloning of the murine ORAI1, ORAI3, and STIM1. Two chromosomal loci were identified for the murine orai2 gene, one containing an intronless gene and a second locus that gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Northern blots revealed a prominent expression of the ORAI2 variants in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appeared to be near ubiquitously expressed in mice tissues. Specific antibodies detected ORAI2 in RBL 2H3 but not in HEK 293 cells, whereas both cell lines appeared to express ORAI1 and STIM1 proteins. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhanced substantially CRAC current densities in HEK 293 but were ineffective in RBL 2H3 cells, whereas ORAI1 strongly amplified CRAC currents in both cell lines. Thus, the capability of ORAI2 variants to form CRAC channels depends strongly on the cell background. Additionally, CRAC channels formed by ORAI2S were strongly sensitive to inactivation by internal Ca(2+). When co-expressed with STIM1 and ORAI1, ORAI2S apparently plays a negative dominant role in the formation of CRAC channels.

The Ca(2+) homeostasis within cells controls a diversity of cellular processes including gene transcription, proliferation and apoptosis. Perturbance of Ca(2+) signaling may induce deregulation of cell proliferation and suppression of cell death providing the basis for cancer development. In human prostate cancer, a correlation between the mRNA expression of the Ca(2+) channel TRPV6 and the staging of the cancer has been described. We have analyzed the influence of TRPV6 on cell proliferation within HEK-293 cells. We show that TRPV6 increases cell proliferation of HEK-293 cells in a Ca(2+) dependent manner. The increased proliferation correlates with slightly increased intracellular Ca(2+) levels without interfering with the intrinsic Ca(2+) dependence of HEK-293 cell proliferation. Low doses of econazole inhibit both, TRPV6 dependent Ca(2+) signals and cell proliferation while BTP2, a potent inhibitor of Ca(2+) signals and cell proliferation in T-cells, neither influences TRPV6 dependent Ca(2+) signals nor cell proliferation of HEK-293 cells. Our data demonstrate that TRPV6 increases the rate of Ca(2+) dependent cell proliferation which is a prerequisite for its potential role in tumor progression.

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

Life expectancy for patients suffering from prostate cancer is inversely correlated with the degree of extraprostatic metastasis. In order to find pharmacological tools to treat this aggressive growth it is important to define targets whose expression not only correlates with the malignancy of the cancerous cells, but that are also amenable to pharmacological intervention. In this review, we would like to focus on the potential role of a distinct class of ion channels that may be involved in this process., (Copyright 2004 Elsevier Inc.)

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.

Transient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly.

Calcium (Ca2+) is an ubiquitous intracellular signal that is responsible for a plethora of cellular processes including fertilization, secretion, contraction, neuronal signaling and learning. In addition, changes in intracellular Ca2+ have been known to influence cell proliferation and differentiation for more than three decades. Recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels which respond to many different modes of stimulation both from within and outside the cell may be a primary mode of cation and Ca2+ entry into cells and may have roles in growth control. Moreover, changes in the expression of these channels may contribute to certain cancers. In the following, recent results concerning the expression and function of members of this family of ion channels are summarized.

TRP proteins, in most cases, provide localized Ca2+ increases for spatially defined signal transduction processes. They are activated by as yet unclear mechanisms, many involving the complex phospholipase C and phosphatidylinositol pathways. In mouse endothelial cells at least seven TRPs are expressed, including TRPC1, TRPC2, TRPC3, TRPC4, TRPC6, TRPV4 and TRPM4. As shown previously, TRPC4 is an indispensable component of agonist-induced Ca2+ entry channels in native endothelial cells which essentially contributes to agonist-induced vessel relaxation and microvascular endothelial permeability, although, it is still open, whether TRPC4 acts as channel-forming subunit and/or essential constituent for channel activation. Utilizing the mouse model is one way to address this question and to provide novel insights for the biological functions of TRPC4. Here we review recent results on heterologously expressed TRPC4 and summarize what is known on the phenotype of the TRPC4-/- mice generated in our laboratory.

Neuronal dendrites have been shown to actively contribute to synaptic information transfer through the Ca2+-dependent release of neurotransmitter, although the underlying mechanisms remain elusive. This study shows that the increase in dendritic gamma-aminobutyric acid (GABA) release from thalamic interneurons mediated by the activation of 5-hydroxytryptamine type 2 receptors requires Ca2+ entry that does not involve Ca2+ release nor voltage-gated Ca2+ channels in the plasma membrane but that is critically dependent on the transient receptor potential (TRP) protein TRPC4. These data ascribe a functional role of agonist-activated TRP channels to the release of transmitters from dendrites, thereby indicating a principle underlying synaptic interactions in the brain.

The contribution of endogenous and recombinant transient receptor potential vanilloid type 6 (TRPV6) channels to Ca2+ entry across the plasma membrane was studied in the human lymph node prostate cancer cell line (LNCaP). LNCaP cells do express the TRPV6 gene, and Ca2+ entry currents in these cells were detected after active and passive Ca2+ store depletion by intracellular application of inositol 1,4,5-trisphosphate, Ca2+ chelators, and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. This store-operated Ca2+ current (ISOC) had biophysical properties similar to those of the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukemia cells such as the activation mechanism, inward rectification, and Ca2+ selectivity. These properties are also shared by the Ca2+-sensing Ca2+ current (ITRPV6) recorded after heterologous expression of TRPV6 cDNA in human embryonic kidney and rat basophilic leukemia cells (Bödding, M., Wissenbach, U., Flockerzi, V. (2002) J. Biol. Chem. 277, 36656-36664). TRPV6 cDNA transfection of LNCaP cells restored recombinant ITRPV6, which can be distinguished from ISOC by the mechanism of activation, the voltage dependence of monovalent currents in the absence of external divalent cations, and the changes in Ca2+ current densities due to different membrane potentials. In addition, ISOC was not affected by antiandrogen or 1,25-dihydroxyvitamin D3 treatment of LNCaP cells, which up-regulates TRPV6 gene expression, or by androgen treatment, which has the opposite effect. Therefore, native channels responsible for ISOC are different from those for recombinant ITRPV6 and do not appear to be affected if one of their assumed subunits, TRPV6, is up- or down-regulated, suggesting a rather rigid subunit composition in vivo.

Members of the TRP superfamily of cation channels have homeostatic and regulatory functions in cells and changes in their expression may contribute to malignant growth. Previously, we have demonstrated that the gene of the Ca2+-selective cation channel CaT-L or TRPV6 is not expressed in benign prostate tissues including benign prostate hyperplasia, but is upregulated in prostate cancer. Here, we report on the differential expression of TRPV6 mRNA in prostate tissue obtained from 140 patients with prostate cancer. Using in situ hybridization, TRPV6 transcripts were undetectable in benign prostate tissue, high-grade prostatic intraepithelial neoplasia (n=57), incidental adenocarcinoma and all tumors less than 2.3 cubic centimeter (cc). In prostatectomy specimens from 97 clinically organ-confined tumors, TRPV6 expression correlated significantly with the Gleason score (P=0.032), pathological stage (P<0.001) and extraprostatic extension (P=0.025). Lymph node metastasis (n=17) and androgen-insensitive tumors (n=27) revealed TRPV6 expression in 63 and 67% of cases, respectively. The latter, however, revealed markedly and significantly decreased levels when compared with untreated tumors (P=0.044). In summary, the data demonstrate that TRPV6 expression is associated with prostate cancer progression. Accordingly, TRPV6 represents a prognostic marker and, as a plasma membrane Ca2+ channel, a promising target for new therapeutic strategies to treat advanced prostate cancer.

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.

The mouse TRPV6 gene is localized on chromosome 6 and extends over 15.66kb. The encoded protein comprises 727 amino acid residues with a calculated relative molecular mass of 83,210Da. TRPV6 is glycosylated and both variants, the glycosylated and the de-glycosylated proteins, are recognized by various polyclonal and monoclonal antibodies, which were raised against TRPV6. Like human TRPV6, mouse TRPV6 binds calmodulin in the presence, but not in the absence of Ca2+. TRPV6 is abundantly expressed in mouse pancreas and placenta, and to a much lesser extend in mouse stomach and kidney. No transcript expression was detected in poly(A)+RNA isolated from heart, brain, intestine, esophagus or aortic endothelial cells.

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

Store-operated Ca(++) entry (SOCE) is thought to comprise the major pathway for Ca(++) entry in platelets. Recently, a number of transient receptor potential (TRP) proteins, which have been divided into 3 groups (TRPC, TRPM, and TRPV), have been suggested as SOCE channels. We report the expression and function of TRPC proteins in human platelets. TRPC6 is found at high levels and TRPC1 at low levels. Using purified plasma (PM) and intracellular membranes (IM), TRPC6 is found in the PM, but TRPC1 is localized to the IM. Using Fura-2-loaded platelets, we report that, in line with TRPC6 expression, 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the entry of Ca(++) and Ba(2+) independently of protein kinase C. Thrombin also induced the entry of Ca(++) and Ba(2+), but thapsigargin, which depletes the stores, induced the entry of only Ca(++). Thus, thrombin activated TRPC6 via a SOCE-independent mechanism. In phosphorylation studies, we report that neither TRPC6 nor TRPC1 was a substrate for tyrosine kinases. TRPC6 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK) and associated with other cAMP-PK substrates. TRPC1 was not phosphorylated by cAMP-PK but also associated with other substrates. Activation of cAMP-PK inhibited Ca(++) but not Ba(2+) entry induced by thrombin and neither Ca(++) nor Ba(2+) entry stimulated by OAG. These results suggest that TRPC6 is a SOCE-independent, nonselective cation entry channel stimulated by thrombin and OAG. TRPC6 is a substrate for cAMP-PK, although phosphorylation appears to not affect cation permeation. TRPC1 is located in IM, suggesting a role at the level of the stores.

The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca(2+) transporter-like protein and Ca(2+) transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca(2+)-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca(2+) entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca(2+) concentration; lowering the intracellular free Ca(2+) concentration results in Ca(2+) influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca(2+) by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca(2+) release-activated Ca(2+) current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca(2+)-sensing Ca(2+) channel independently of procedures known to deplete Ca(2+) stores.

We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4(-/-) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4(-/-) LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 micromol/L). In TRPC4(-/-) LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4(-/-) endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (K(f,c)), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4(-/-); La3+ (1 micromol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4(-/-). These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.

TRP cation channels display an extraordinary assortment of selectivities and activation mechanisms, some of which represent previously unrecognized modes for regulating ion channels. Moreover, the biological roles of TRP channels appear to be equally diverse and range from roles in pain perception to male aggression.