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Abstract Tracheal tuft cells shape immune responses in the airways. While some of these effects have been attributed to differential release of either acetylcholine, leukotriene C4 and/or interleukin-25 depending on the activating stimuli, tuft cell-dependent mechanisms underlying the recruitment and activation of immune cells are incompletely understood. Here we show that Pseudomonas aeruginosa infection activates mouse tuft cells, which release ATP via pannexin 1 channels. Taste signaling through the Trpm5 channel is essential for bacterial tuft cell activation and ATP release. We demonstrate that activated tuft cells recruit dendritic cells to the trachea and lung. ATP released by tuft cells initiates dendritic cell activation, phagocytosis and migration. Tuft cell stimulation also involves an adaptive immune response through recruitment of IL-17A secreting T helper cells. Collectively, the results provide a molecular framework defining tuft cell dependent regulation of both innate and adaptive immune responses in the airways to combat bacterial infection.

Abstract Background The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. Results We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. Conclusions We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes.

Abstract The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA–target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson’s disease (PD). After performing 13,536 transfections, we validated 442 of the 705 putative MTIs (62,7%) through dual luciferase reporter assays. These analyses increased the number of validated MTIs by at least 2.1-fold for miR-133b and by a maximum of 24.3-fold for miR-873-5p. Our study contributes to the experimental capture of miRNA targetomes by addressing i) the ratio of experimentally verified MTIs to predicted MTIs, ii) the sizes of disease-related miRNA targetomes, and iii) the density of MTI networks. A web service to support the analyses on the MTI level is available online ( https://ccb-web.cs.uni-saarland.de/utr-seremato ), and all the data have been added to the miRATBase database ( https://ccb-web.cs.uni-saarland.de/miratbase ).

Abstract Background Airway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive. Methods We performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed. Results Tuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance. Conclusions We were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.

Abstract Among the concepts in biology that are widely taken granted is a potentiated cooperative effect of multiple miRNAs on the same target. This strong hypothesis contrasts insufficient experimental evidence. The quantity as well as the quality of required side constraints of cooperative binding remain largely hidden. For miR-21-5p and miR-155-5p, two commonly investigated regulators across diseases, we selected 15 joint target genes. These were chosen to represent various neighboring 3′UTR binding site constellations, partially exceeding the distance rules that have been established for over a decade. We identified different cooperative scenarios with the binding of one miRNA enhancing the binding effects of the other miRNA and vice versa. Using both, reporter assays and whole proteome analyses, we observed these cooperative miRNA effects for genes that bear 3′UTR binding sites at distances greater than the previously defined limits. Astonishingly, the experiments provide even stronger evidence for cooperative miRNA effects than originally postulated. In the light of these findings the definition of targetomes specified for single miRNAs need to be refined by a concept that acknowledges the cooperative effects of miRNAs.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

To identify TRPV6 expression in the whole mouse with a cellular resolution we took advantage of TRPV6-IRES-Cre knock-in mice crossed with the enhanced ROSA26-τGFP reporter line. In the resulting TRPV6-IC/eR26-τGFP animals, TRPV6-expressing cells are labeled with τGFP. Data were collected from organs prepared from fixed experimental adult and juvenile TRPV6-IC/eR26τGFP and Cre-negative eR26-τGFP control animals of both sexes. Organ cryosections from each age and sex were stained for GFP and imaged with a slide scanner. Here, we describe reporter gene expression in a large number of tissues. We also document the absence of τGFP signal in the corresponding Cre-negative control tissues, including controls for the TRPV6 expression data described in [1]. The data reported here and in [1] constitute the TRPV6 expression atlas for the mouse. Our data offer a wealth of information to enable investigation of the functional role of TRPV6 channels in different tissues.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice. • We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution. • We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice.

Summary: Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7−/− embryos are able to hatch from the zona pellucida, no expansion of Trpm7−/− trophoblast cells can be observed, and Trpm7−/− embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7−/− trophoblast stem cells and the ability of Trpm7−/− ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.

Mucociliary clearance is a primary defence mechanism of the airways consisting of two components, ciliary beating and transepithelial ion transport (ISC). Specialised chemosensory cholinergic epithelial cells, named brush cells (BC), are involved in regulating various physiological and immunological processes. However, it remains unclear if BC influence ISC. In murine tracheae, denatonium, a taste receptor agonist, reduced basal ISC in a concentration-dependent manner (EC50 397 µM). The inhibition of bitter taste signalling components with gallein (Gβγ subunits), U73122 (phospholipase C), 2-APB (IP3-receptors) or with TPPO (Trpm5, transient receptor potential-melastatin 5 channel) reduced the denatonium effect. Supportively, the ISC was also diminished in Trpm5−/− mice. Mecamylamine (nicotinic acetylcholine receptor, nAChR, inhibitor) and amiloride (epithelial sodium channel, ENaC, antagonist) decreased the denatonium effect. Additionally, the inhibition of Gα subunits (pertussis toxin) reduced the denatonium effect, while an inhibition of phosphodiesterase (IBMX) increased and of adenylate cyclase (forskolin) reversed the denatonium effect. The cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh172 and the KCNQ1 potassium channel antagonist chromanol 293B both reduced the denatonium effect. Thus, denatonium reduces ISC via the canonical bitter taste signalling cascade leading to the Trpm5-dependent nAChR-mediated inhibition of ENaC as well as Gα signalling leading to a reduction in cAMP-dependent ISC. Therefore, BC activation contributes to the regulation of fluid homeostasis.

Summary: Multiple sclerosis (MS) is a demyelinating disease caused by an auto-reactive immune system. Recent studies also demonstrated synapse dysfunctions in MS patients and MS mouse models. We previously observed decreased synaptic vesicle exocytosis in photoreceptor synapses in the EAE mouse model of MS at an early, preclinical stage. In the present study, we analyzed whether synaptic defects are associated with altered presynaptic Ca2+ signaling. Using high-resolution immunolabeling, we found a reduced signal intensity of Cav-channels and RIM2 at active zones in early, preclinical EAE. In line with these morphological alterations, depolarization-evoked increases of presynaptic Ca2+ were significantly smaller. In contrast, basal presynaptic Ca2+ was elevated. We observed a decreased expression of Na+/K+-ATPase and plasma membrane Ca2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca2+. Thus, complex Ca2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE.

Summary: Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N and C termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis, and cancer. The TRPV6 function and regulation remain poorly understood. Here we show that the TRPV6 intramolecular S4-S5 linker to C-terminal TRP helix (L/C) and N-terminal pre-S1 helix to TRP helix (N/C) interactions, mediated by Arg470:Trp593 and Trp321:Ile597 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on the L/C interaction but not reversely. Three cationic residues in S5 or C terminus are involved in binding PIP2 to suppress both interactions thereby activating TRPV6. This study reveals “PIP2 - intramolecular interactions” regulatory mechanism of TRPV6 activation-autoinhibition, which will help elucidating the corresponding mechanisms in other TRP channels.

The new psychoactive substances (NPS) market continues to be very dynamic. A large number of compounds belonging to diverse chemical groups continue to emerge. This makes their detection in biological samples challenging for clinical and forensic toxicologists. Knowledge of the metabolic fate of NPS is crucial for developing comprehensive screening procedures. As human studies are not feasible due to ethical concerns, the current study aimed to compare the NPS' metabolic pattern in incubations with pooled human liver S9 fraction (pHLS9), human liver HepaRG cells, and zebrafish larvae. The latter model was recently shown to be a promising preclinical surrogate for human hepatic metabolism of a synthetic cannabinoid. However, studies concerning other NPS classes are still missing and therefore an amphetamine-based N-methoxybenzyl (NBOMe) compound, a synthetic cathinone, a pyrrolidinophenone analog, a lysergamide, as well as another synthetic cannabinoid were included in the current study. Liquid chromatography coupled to Orbitrap-based high-resolution tandem mass spectrometry was used to analyze metabolic data. Zebrafish larvae were found to produce the highest number of phase I but also phase II metabolites (79 metabolites in total), followed by HepaRG cells (66 metabolites). Incubations with pHLS9 produced the least metabolites (57 metabolites). Furthermore, the involvement of monooxygenases and esterases in the metabolic phase I transformations of 4F-MDMB-BINACA was elucidated using single-enzyme incubations. Several cytochrome P450 (CYP) isozymes were shown to contribute, and CYP3A5 was involved in all CYP-catalyzed reactions, while amide and ester hydrolysis were catalyzed by the human carboxylesterase (hCES) isoforms hCES1b and/or hCES1c. Finally, metabolites were compared to those present in human biosamples if data were available. Overall, the metabolic patterns in HepaRG cells provided the worst overlap with that in human biosamples. Zebrafish larvae experiments agreed best with data found in human plasma and urine analysis. The current study underlines the potential of zebrafish larvae as a tool for elucidating the toxicokinetics of NPS in the future.

Summary: By vacuolar patch-clamp and Ca2+ imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca2+ and inhibited by Ca2+ from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca2+ (Hill coefficient, 1.5), suggesting that it may accommodate a Ca2+ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of 45Ca2+. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca2+ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca2+ binding. : Structural Biology; Protein Structure Aspects; Biophysics Subject Areas: Structural Biology, Protein Structure Aspects, Biophysics

Background & Aims: 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 is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ 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 Cav1.3 calcium channels. Femurs were analyzed by using micro–computed tomography and histology. Results: Net TRPV6-mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Cav1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Cav1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca2+ absorption in the ileum, and increased expression of renal Ca2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Cav1.3 expression. Conclusions: We have detailed novel pathways contributing to transcellular Ca2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance early in life. Keywords: Calcium Channel, Pediatric, Bone, Development

Abstract 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.

Summary: A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T’s 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T’s aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies. : A mutation in TRPM4 (A432T) is linked to life-threatening cardiac conduction disturbances in patients. Using a combination of biophysical techniques, Xian et al. demonstrate that calcium-dependent deactivation between heartbeats is aberrant and highlights the etiology of human cardiac channelopathies. These findings offer putative new pharmacological targets for disease management in human patients. Keywords: TRPM4, calcium, membrane current, cardiac arrhythmia, inherited human disease, mutation, flash photolysis, patch clamp, molecular modeling, disease mechanism

Mutations in the cation channel PKD2 cause human autosomal dominant polycystic kidney disease but its channel function and gating mechanism are poorly understood. Here authors study PKD2 using electrophysiology and cryo-EM, which identifies hydrophobic gates and proposes a gating mechanism for PKD2.

Summary: Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

Summary: Voltage-gated calcium channels (Cavs) are major Ca2+ 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 Ca2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP3) formation. This effect requires the SH3-HOOK domain of Cavβ3, includes physical β3/IP3 receptor interaction, and prevails when agonist-induced IP3 formation is bypassed by photolysis of caged IP3. In agreement with β3 acting as a brake on Ca2+ 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 Ca2+ toxicity, cytoplasmic Ca2+ signals must be tightly controlled. The described function of β3, unrelated to its function as a Cav subunit, adds to this tight control. : Belkacemi et al. show that the calcium channel subunit Cavβ3 binds to the IP3R and desensitizes cells to low IP3 levels, influencing fibroblast migration and collagen secretion. Removal of the Cavβ3 protein in mice results in faster skin wound healing. Keywords: Ca2+ signaling, Cavβ3, cell migration, IP3 receptor, wound healing, Cavβ2, Cavβ3 KO, IP3 binding, voltage-gated Ca2+ channel, Ca2+ release

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.

Ca2+ release-activated Ca2+(CRAC) channels are essential for protective immunity, but the immunological functions of the three ORAI homologues that form CRAC channels are unclear. Here the authors show that ORAI1 and ORAI2 form heteromeric CRAC channels, which fine-tune T cell activation and immune responses.

Pathological cardiac remodeling correlates with chronic neurohumoral stimulation and abnormal Ca2+ signaling in cardiomyocytes. Store-operated calcium entry (SOCE) has been described in adult and neonatal murine cardiomyocytes, and Orai1 proteins act as crucial ion-conducting constituents of this calcium entry pathway that can be engaged not only by passive Ca2+ store depletion but also by neurohumoral stimuli such as angiotensin-II. In this study, we, therefore, analyzed the consequences of Orai1 deletion for cardiomyocyte hypertrophy in neonatal and adult cardiomyocytes as well as for other features of pathological cardiac remodeling including cardiac contractile function in vivo. Cellular hypertrophy induced by angiotensin-II in embryonic cardiomyocytes from Orai1-deficient mice was blunted in comparison to cells from litter-matched control mice. Due to lethality of mice with ubiquitous Orai1 deficiency and to selectively analyze the role of Orai1 in adult cardiomyocytes, we generated a cardiomyocyte-specific and temporally inducible Orai1 knockout mouse line (Orai1CM–KO). Analysis of cardiac contractility by pressure-volume loops under basal conditions and of cardiac histology did not reveal differences between Orai1CM–KO mice and controls. Moreover, deletion of Orai1 in cardiomyocytes in adult mice did not protect them from angiotensin-II-induced cardiac remodeling, but cardiomyocyte cross-sectional area and cardiac fibrosis were enhanced. These alterations in the absence of Orai1 go along with blunted angiotensin-II-induced upregulation of the expression of Myoz2 and a lack of rise in angiotensin-II-induced STIM1 and Orai3 expression. In contrast to embryonic cardiomyocytes, where Orai1 contributes to the development of cellular hypertrophy, the results obtained from deletion of Orai1 in the adult myocardium reveal a protective function of Orai1 against the development of angiotensin-II-induced cardiac remodeling, possibly involving signaling via Orai3/STIM1-calcineurin-NFAT related pathways.

The original version of this Article contained an error in the spelling of the author David Bulkley, which was incorrectly given as David Bulkey. This has now been corrected in both the PDF and HTML versions of the Article.

Summary Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.