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Voltage-gated Ca2+ (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 (IP3). Similarly, Cavβ3 could affect cytosolic [Ca2+] in pancreatic β-cells. Here, 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 Ca2+-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO 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 IP3-dependent Ca2+ release and the frequency of Ca2+-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca2+-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca2+-selective and has been implicated in mediating transcellular Ca2+ transport and thus maintaining the Ca2+ 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.

Significance The strengthening of heart contraction by epinephrine (adrenaline) and norepinephrine starts with the activation of β-adrenergic receptors and culminates in a protein kinase A–mediated increase in Ca 2+ influx through the voltage-gated Ca 2+ channel, Ca V 1.2, into cardiomyocytes. Many crucial molecular details of this vital physiological regulation remained enigmatic for decades, not the least owing to the difficulty of reconstituting the regulation in model cells. Capitalizing on the recent discovery of the central role of the Ca V 1.2-associated protein, Rad, we present a report of full reconstitution of the β-AR–Ca V 1.2 cascade in a model system, the Xenopus oocyte, and investigate crucial aspects of regulation such as the roles of auxiliary subunits and diverse forms of the channel protein.

IntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete β-AR CaV1.2 modulation cascade in a heterologous expression system.ObjectiveOur primary goal was to heterologously reconstitute the complete β-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2.Methods and ResultsWe utilized theXenopusoocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and β1-AR or β2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate β-AR. Whole-cell Ba2+currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of α1Cand for the CaVβ subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either β1-AR or β2-AR. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR.ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of CaV1.2 activity, crucially depends on CaVβ subunit, and is differently regulated by β1-AR and β2-AR. The reconstitution of the full β-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.

Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6Ala616 or Met617 or TRPV5Ala576 or Met577, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6Ala616-Met617 and TRPV5Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4Iso715, TRPC4Iso617, and TRPM8Val976. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+–CaM required the cytoplasmic amino acid stretch E33–Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+–CaM.

The protein Flower mediates the calcium-dependence of synaptic vesicle endocytosis in Drosophila. Chang et al. show that Flower is predominantly localized on intracellular vesicles that move to the synapse upon target cell contact and that Flower is required for efficient endocytosis of cytotoxic granules in T cells., 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.

Transient receptor potential (TRP) ion channels regulate fundamental biological processes throughout the body. TRP channel dysfunction has been causally linked to a number of disease states and thus establishes these channels as promising therapeutic targets. In order to dissect the physiological role of individual TRP channels in specific tissues, a detailed understanding of the expression pattern of the different TRP channels throughout the organism is essential. We provide an overview of recent efforts to generate novel TRP channel reporter mouse strains for all 28 TRP channels encoded in the mouse genome to understand expression of these channels with a single-cell resolution in an organism-wide manner. The reporter mice will enable both the visualization and manipulation of all primary TRP channel-expressing cells allowing an unprecedented wealth in variety to investigate TRP channel function in vivo. As proof of principle, we provide preliminary results documenting TRPM5 expression throughout the entire body of juvenile and adult mice.

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high‐resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5 ‐triple‐knockout ( Trpc1/4/5 −/− ) mice, lacking any TRPC1‐, TRPC4‐, or TRPC5‐containing channels, action potential‐triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo , Trpc1/4/5 −/− mice displayed impaired cross‐frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5 −/− animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, reduces the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5–τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity., Transient receptor potential (TRP) proteins can form non-selective cation channels, but their role in synaptic transmission is poorly understood. This study shows that calcium-permeable TRPC channels provide an additional calcium entry pathway at presynaptic sites and are efficient regulators of synaptic strength and plasticity.

β-adrenergic stimulation enhances Ca(2+) currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signaling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the posttranslational proteolytic truncation of distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by 40%(rabbit α1C)to 130% (mouse α1C). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for PKA effect. CaV β2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural "scaffold" comprising the interacting cytosolic N- and C-termini of α1C .

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor β (TGF-β1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-β1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-β1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.

New psychoactive substances (NPS) are an emerging topic amongst abused compounds. New varieties appear constantly on the market, without any knowledge about their toxicodynamic and/or -kinetic properties and knowledge of their metabolism is crucial for the development of analytical methods employed for their detection. Controlled human studies would of course be best suited but due to ethical reasons and lack of preclinical safety data, they are usually not available. Often, in vitro models are used to evaluate similarities to human in vivo hepatic phase I and II metabolism and systems explored include primary human hepatocytes, pooled human S9 fraction, and HepaRG, a human hepatic cell line. All these in vitro models have considerable limitations and drug distribution, reabsorption, enterohepatic circulation, and renal elimination cannot be studied. In the recent years, zebrafish (Danio rerio) larvae (embryos) were discussed as a potential in vivo model to overcome these limitations. To date, no studies demonstrating its suitability for studying NPS metabolism in the context of analytical toxicology are available. The aim of this study was to elucidate whether zebrafish larvae can serve as a surrogate for human hepatic metabolism of NPS to develop toxicological screening procedures. Here, we used methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (7'N-5F-ADB), a new synthetic cannabinoid, whose human metabolism was recently described in the literature, as a model compound to evaluate zebrafish larvae as a new tool for metabolism studies. Different conditions for zebrafish larvae and HepaRG protocols were tested. As zebrafish larvae and HepaRG cell incubations provided the highest number of metabolites and the most authentic spectrum of human metabolites. The most suitable larvae protocol was the incubation via medium and the analysis of the extracted zebrafish larvae. The zebrafish larvae model might be a promising preclinical surrogate for human hepatic metabolism of NPS.

Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto‐immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well‐validated mouse models of optic neuritis that ribbon synapses in the myelin‐free retina are targeted by an auto‐reactive immune system even before alterations in the optic nerve have developed. The auto‐immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.

Objectives: Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults, after peak bone mass has been reached, but are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption across the small intestine which facilitate a positive calcium balance during growth.Methods: We used wildtype, Cav1.3 knockout and Trpv6 mutant mice. Expression of small intestinal and renal calcium transport genes was assessed using quantitative PCR. Net transcellular 45-calcium flux across intestinal segments was measured in Ussing chambers. Femurs we analyzed using micro-CT and histology.Results: Significant TRPV6 mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but occurred post-weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum and ileum at P14 but not 2 months. TRPV6 and Cav1.3 are necessary for this jejunal absorption and Cav1.3 appears to mediate absorption across the ileum although compensation is present in knockout pups. Knockout of Cav1.3 induces a compensatory increase in renal Ca2+ reabsorption in P14 mice although these pups have increased growth plate thickness suggesting delayed bone mineralization.Conclusions: This work provides molecular details of how the small intestine facilitates increased demand for Ca2+ early in life to meet the requirements of growth and highlights the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance.Funding Sources: This work is funded by grants from the Women and Children's Health Research Institute, supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology. MRB is supported by a Vanier Canada Graduate Scholarship, Alberta Innovates Clinician Fellowship and an NSERC Michael Smith Foreign Study Supplement. H. Dimke is funded by the Danish Medical Research Council. Work at UdS was funded by Deutsche Forschungsgemeinschaft (DFG) by IRTG1830 (to JE, VF), Sonderforschungsbereich (SFB) 894 (to JE, PW) and SFB TRR152 (to VF).

Purpose To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON). Methods C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials. Results All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant. Conclusions The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

The Ca2+-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca2+-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca2+ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca2+ entry.

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.

We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+)-sensor, and Orai1, a Ca(2+) selective channel, in regulating Ca(2+) entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca(2+) entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca(2+) entry secondary to depletion of ER Ca(2+) stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1or Orai3) or expression of the dominant-negative STIM1(K684E-K685E) mutant in ECs also suppressed Ca(2+) entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca(2+) entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca(2+) entry indicating the requirement for TRPC4 in mediating store-operated Ca(2+) entry. Moreover, expression of the dominant-negative Orai1(R91W) mutant or Orai3(E81W) mutant in WT-ECs failed to prevent thrombin-induced Ca(2+) entry. In contrast, expression of the dominant-negative TRPC4(EE647-648KK) mutant in WT-ECs markedly reduced thrombin-induced Ca(2+) entry. In ECs expressing YFP-STIM1, ER-store Ca(2+) depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca(2+) sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca(2+) stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca(2+)-entry channels, which are essential for mediating Ca(2+) entry-dependent disruption of the endothelial barrier.

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila Norp A phospholipase C which mediates rhabdomeric phototransduction. The Plcb4^(−/−) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary β subunit (Ca(V)β2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)β2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)β2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)β2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)β2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)β proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

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.

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.

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVbeta2 proteins are the major CaVbeta components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVbeta3 proteins are also detectable. At least two CaVbeta2 variants of approximately 68 and approximately 72 kDa are expressed. To identify the underlying CaVbeta2 variants, cDNA libraries were constructed from poly(A)(+) RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVbeta2 cDNA clones coding for four types of CaVbeta2 proteins only differing in their 5' sequences. CaVbeta2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVbeta2 protein pattern in vitro. Significant L-type Ca(2+) currents (I(Ca)) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVbeta2. Current kinetics were determined by the type of CaVbeta2 protein, with the approximately 72-kDa CaVbeta2a-N1 shifting the activation of I(Ca) significantly to depolarizing potentials compared with the other CaVbeta2 variants. Inactivation of I(Ca) was accelerated by CaVbeta2a-N1 and -N4, which also lead to slower activation compared with CaVbeta2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVbeta2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVbeta2a-N1-induced changes of I(Ca) kinetics might be essential in embryonic heart.

Background & Aims Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. Methods We created trpc4 -, trpc6 -, and trpc4/trpc6 -gene–deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. Results In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI CAT ). The residual mI CAT depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI CAT channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. Conclusions In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI CAT . They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca 2+ -influx and contraction, and thereby accelerate small intestinal motility in vivo.

SummaryIn the mammalian central nervous system, slow synaptic excitation involves the activation of metabotropic glutamate receptors (mGluRs). It has been proposed that C1-type transient receptor potential (TRPC1) channels underlie this synaptic excitation, but our analysis of TRPC1-deficient mice does not support this hypothesis. Here, we show unambiguously that it is TRPC3 that is needed for mGluR-dependent synaptic signaling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells. In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+ release signals from intracellular stores are unchanged. Importantly, TRPC3 knockout mice exhibit an impaired walking behavior. Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

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.

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 (CaVγ), 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.

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.

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin–NFAT pathway and the development of pathological hypertrophy. Electronic supplementary material The online version of this article (doi:10.1007/s00395-015-0501-x) contains supplementary material, which is available to authorized users.

TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.

Cardiac myocyte contraction depends on transmembrane L-type Ca 2+ currents and the ensuing release of Ca 2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca 2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Ca v β 2 gene, which encodes for the predominant ancillary β subunit of cardiac Ca 2+ channels, resulted in diminished L-type Ca 2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Ca v β 2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Ca v β 2 proteins in the vasculature. Partial rescue of the Ca 2+ channel currents by a Ca 2+ channel agonist significantly postponed embryonic death in Ca v β 2 −/− mice. Taken together, these data strongly support the essential role of L-type Ca 2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

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.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.

International audience; Transient receptor potential channel subfamily M member 5 (TRPM5) is an important downstream signaling component in a subset of taste receptor cells making it a potential target for taste modulation. Interestingly, TRPM5 has been detected in extra-oral tissues; however, the function of extra-gustatory TRPM5-expressing cells is less well understood. To facilitate visualization and manipulation of TRPM5-expressing cells in mice, we generated a Cre knock-in TRPM5 allele by homologous recombination. We then used the novel TRPM5-IRES-Cre mouse strain to report TRPM5 expression by activating a tau GFP transgene. To confirm faithful coexpression of tau GFP and TRPM5 we generated and validated a new anti-TRPM5 antiserum enabling us to analyze acute TRPM5 protein expression. tau GFP cells were found in taste bud cells of the vallate, foliate, and fungiform papillae as well as in the palate. We also detected TRPM5 expression in several other tissues such as in the septal organ of Masera. Interestingly, in the olfactory epithelium of adult mice acute TRPM5 expression was detected in only one (short microvillar cells) of two cell populations previously reported to express TRPM5. The TRPM5-IC mouse strain described here represents a novel genetic tool and will facilitate the study and tissue-specific manipulation of TRPM5-expressing cells in vivo.

We investigated the possibility that the TRPC gene family of putative store-operated Ca 2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca 2+ 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-NH 2 ) resulted in a prolonged Ca 2+ transient secondary to influx of Ca 2+ . Ca 2+ influx activated by thrombin was blocked by La 3+ (1 μmol/L). In TRPC4 −/− LECs, thrombin or TFLLRNPNDK-NH 2 produced a similar initial increase of intracellular Ca 2+ secondary to Ca 2+ store depletion, but Ca 2+ influx induced by these agonists was drastically reduced. The defect in Ca 2+ 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 −/− ; La 3+ (1 μmol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4 −/− . These results show that TRPC4-dependent Ca 2+ entry in mouse LECs is a key determinant of increased microvascular permeability.

A finely tuned Ca 2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca 2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca 2+ permeation properties suggest an important function in Ca 2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca 2+ -dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

A novel member of the transient receptor potential (Trp) family of ion channels, Trp12, was identified. The Trp12 mRNA is abundantly expressed in mouse kidney and encodes a protein of 871 amino acid residues. Trp12 transfected cells reveal an elevated cytosolic Ca2+ and respond with a further increase of cytosolic Ca2+ to perfusion with hypoosmotic solutions. The human orthologue of murine Trp12 was localized on a genomic clone derived from human chromosome 12. It is composed of 15 translated exons. The intron placement within that primary structure does not correlate with the previously postulated splice sites in transcripts encoding the stretch-inhibitable channel which shares a high degree of amino acid sequence identity with Trp12 and the vanilloid receptor type 1.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

In many non-excitable as well as electrically excitable cells, depletion of intracellular Ca2+ stores after stimulation of G protein coupled receptors or receptor tyrosine kinases is followed by Ca2+ entry across the plasma membrane, a mechanism referred to as capacitative calcium entry (CCE) [Putney, J.W., Cell Calcium 11 (1990) 611-624; Fasolato, C. et al., Trends Pharmacol Sci. 15 (1994) 77-83]. Recently, we reported that bCCE 1, a homologue of the Drosophila protein trp, exhibits the characteristics of CCE channels [Philipp, S. et al., EMBO J. 15 (1996) 6166-6171]. In this study, we report the cloning of a 5' truncated splice variant (bCCE 1delta514) of the full-length bCCE 1. The bCCE 1delta514 cDNA encodes a protein of 486 amino acids with the ATG triplet encoding M514 of bCCE 1 as translation initiation codon and, therefore, comprises two putative transmembrane segments corresponding to the predicted transmembrane segments 5 and 6 of bCCE 1. bCCE 1delta514 transcripts appear to be specifically expressed in the adrenal gland and genome analysis reveals an alternative splice site within an exon of the CCE 1 gene leading to the formation of bCCE 1delta514.

TRPC4 proteins function as Ca(2+) conducting, non-selective cation channels in endothelial, smooth muscle, and neuronal cells. To further characterize the roles of TRPC4 in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. Therefore, a mouse brain cDNA library was searched for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified Trans-activation Response RNA-binding protein 2 (Tarpb2), a protein that recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Tarbp2 was found to bind to the C terminus of TRPC4 and TRPC5 and to modulate agonist-dependent TRPC4-induced Ca(2+) entry. A stretch of basic residues within the Tarbp2 protein is required for these actions. Tarbp2 binding to and modulation of TRPC4 occurs in the presence of endogenously expressed Dicer but is no longer detectable when the Dicer cDNA is overexpressed. Dicer activity in crude cell lysates is increased in the presence of Ca(2+), most probably by Ca(2+)-dependent proteolytic activation of Dicer. Apparently, Tarbp2 binding to TRPC4 promotes changes of cytosolic Ca(2+) and, thereby, leads to a dynamic regulation of Dicer activity, essentially at low endogenous Dicer concentrations.

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca2+itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that β-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVβ2subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca2+-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

The family of transient receptor potential cation channels has received in the last 10 years a tremendous interest because members of this family are involved in a plethora of cell functions and have been identified as causal for many hereditary and acquired diseases. We shortly introduce these channels, summarize nomenclature and chromosomal location of the 28 mammalian Trp genes, and list the available Trp-deficient mouse lines.

TRPs -Truly Remarkable Proteins.- The TRCP Subfamily: TRPC1.- TRPC2.- TRPC3- a Multifunctional Signaling Molecule.- TRPC4 and TRPC4-Containing Channels.- TRPC5.- TRPC6: Physiological Function and Pathophysiological Relevance.- Transient Receptor Potential Canonical 7 (TRPC7).- The TRPV Subfamily: TRPV1.- TRPV2.- TRPV3.- The TRPV4 Channel.- TRPV5, a Ca2+ Channel for the Fine Tuning of Ca2+ Reabsorption.- TRPV6 Channels.- The TRPM Subfamily: TRPM1.- TRPM2.- TRPM3.-TRPM4.- TRPM5.- TRPM6.- TRPM7.- TRPM8.- The TRPA1, TRPML and TRPP Subfamily: TRPA1.- TRPML1: An Ion Channel in the Lysosome.- TRPML2 and Mucolipin Evolution.- TRPML3.- TRPP Subfamily and Polycystin-1 Proteins.