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Competing Interests: Declaration of competing interest The authors declare to have no competing interest.

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients., (© 2024. The Author(s).)

Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca 2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca 2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca 2+ signals of low amplitude. Subsequently, also global Ca 2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca 2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca 2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic., (© 2024. The Author(s).)

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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

Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca 2+ -permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca 2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca 2+ measurements, (2) peri-endo-lysosomal Ca 2+ imaging using genetically encoded Ca 2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca 2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca 2+ imaging by directing Ca 2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca 2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca 2+ imaging., (© 2023. Springer Nature Switzerland AG.)

Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca 2+ and Mg 2+ . Deletion of the Ca 2+ and Mg 2+ 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 Ca 2+ and Mg 2+ 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Blood-brain barrier (BBB) dysfunction is critically involved in determining the extent of several central nervous systems (CNS) pathologies and here in particular neuroinflammatory conditions. Inhibiting BBB breakdown could reduce the level of vasogenic edema and the number of immune cells invading the CNS, thereby counteracting neuronal injury. Transient receptor potential (TRP) channels have an important role as environmental sensors and constitute attractive therapeutic targets that are involved in calcium homeostasis during pathologies of the CNS. Transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable, non-selective cation channel highly expressed in endothelial cells. As it is involved in the regulation of the blood brain barrier permeability and consequently cerebral edema formation, we anticipated a regulatory role of TRPV4 in CNS inflammation and subsequent neuronal damage. Here, we detected an increase in transendothelial resistance in mouse brain microvascular endothelial cells (MbMECs) after treatment with a selective TRPV4 inhibitor. However, this effect was abolished after the addition of IFNγ and TNFα indicating that inflammatory conditions override TRPV4-mediated permeability. Accordingly, we did not observe a protection of Trpv4 -deficient mice when compared to wildtype controls in a preclinical model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), and no differences in infarct sizes following transient middle cerebral artery occlusion (tMCAO), the experimental stroke model, which leads to an acute postischemic inflammatory response. Furthermore, Evans Blue injections did not show differences in alterations of the blood brain barrier (BBB) permeability between genotypes in both animal models. Together, TRPV4 does not regulate brain microvascular endothelial permeability under inflammation., (Copyright © 2020 Rosenkranz, Shaposhnykov, Schnapauff, Epping, Vieira, Heidermann, Schattling, Tsvilovskyy, Liedtke, Meuth, Freichel, Gelderblom and Friese.)

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca 2+ ) signals. Ca 2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca 2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca 2+ uptake and filling of endoplasmic reticulum Ca 2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential., Competing Interests: Declaration of Interests T.G., L.R.-S., F.C., D.W., J.N., A.H., S.H.-B., B.K.F., and A.P. have filed an application for patenting A2B agonist treatment of musculoskeletal disorders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca 2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca 2+ -mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca 2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABA A receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca 2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca 2+ oscillations triggered by spontaneous glutamate release events., (Copyright © 2020 Hermann, Bender, Schumacher, Woo, Shaposhnykov, Rosenkranz, Kuryshev, Meier, Guse, Friese, Freichel and Tsvilovskyy.)

Background & Aims: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca 2+ -selective ion channel, in an international cohort of patients and in mice., Methods: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca 2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6 mut/mut mice., Results: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10 -8 ). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10 -8 ). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10 -9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10 -5 ). TRPV6 mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis., Conclusions: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca 2+ balance in pancreatic cells. TRPV6 regulates Ca 2+ homeostasis and pancreatic inflammation., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in both BMMC and PMC, robust [Ca 2+ ] i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca 2+ ] i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca 2+ oscillations in BMMC and robust [Ca 2+ ] i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca 2+ ] i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca 2+ ] i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca 2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca 2+ ] i rise was reduced by ~20% in PMC from Trpc1/4/6 -/- mice compared to Trpc1/4 -/- littermatched control mice, whereas FcεRI-evoked [Ca 2+ ] i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca 2+ ] i rise in Trpc1/4/6 -/- PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca 2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified., (Copyright © 2020 Tsvilovskyy, Solis-Lopez, Almering, Richter, Birnbaumer, Dietrich and Freichel.)

Mast cells (MCs), which are best known for their detrimental role in patients with allergic diseases, act in a diverse array of physiologic and pathologic functions made possible by the plurality of MC types. Their various developmental avenues and distinct sensitivity to (micro-) environmental conditions convey extensive heterogeneity, resulting in diverse functions. We briefly summarize this heterogeneity, elaborate on molecular determinants that allow MCs to communicate with their environment to fulfill their tasks, discuss the protease repertoire stored in secretory lysosomes, and consider different aspects of MC signaling. Furthermore, we describe key MC governance mechanisms (ie, the high-affinity receptor for IgE [FcεRI]), the stem cell factor receptor KIT, the IL-4 system, and both Ca 2+ - and phosphatase-dependent mechanisms. Finally, we focus on distinct physiologic functions, such as chemotaxis, phagocytosis, host defense, and the regulation of MC functions at the mucosal barriers of the lung, gastrointestinal tract, and skin. A deeper knowledge of the pleiotropic functions of MC mediators, as well as the molecular processes of MC regulation and communication, should enable us to promote beneficial MC traits in physiology and suppress detrimental MC functions in patients with disease., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Methylglyoxal (MG) is a by-product of glucose metabolism and its accumulation has been linked to the development of diabetic complications such as retinopathy and nephropathy by affecting multiple signalling pathways. However, its influence on the intracellular Ca 2+ homeostasis and particularly Ca 2+ entry, which has been reported to be mediated via TRPA1 channels in DRG neurons, has not been studied in much detail in other cell types. In this study, we report the consequences of acute and long-term MG application on intracellular Ca 2+ levels in endothelial cells. We showed that acute MG application doesn't evoke any instantaneous changes in the intracellular Ca 2+ concentration in immortalized mouse cardiac endothelial cells (MCECs) and murine microvascular endothelial cells (muMECs). In contrast, an MG-induced rise in intracellular Ca 2+ level was observed in primary mouse mesangial cells within 30 s, indicating that the modulation of Ca 2+ homeostasis by MG is strictly cell type specific. The formation of the MG-derived advanced glycation end product (AGE) MG-H1 was found to be time and concentration-dependent in MCECs. Likewise, MG pre-incubation for 6 h increased the angiotensin II-evoked Ca 2+ entry in MCECs and muMECs which was abrogated by inhibition of Calcium release activated calcium (CRAC) channels with GSK-7975A, but unaffected by an inhibitor specific to TRPA1 channels. Quantitative PCR analysis revealed that MG pre-treatment did not affect expression of the genes encoding the angiotensin receptors AT1R (Agtr 1a & Agtr 1b), Trpa1 nor Orai1, Orai2, Orai3, Stim1, Stim2 and Saraf which operate as constituents or regulators of CRAC channels and store-operated Ca 2+ entry (SOCE) in other cell types. Together, our results show that long-term MG stimulation leads to the formation of glycation end products, which facilitates the agonist-evoked Ca 2+ entry in endothelial cells, and this could be a new pathway that might lead to MG-evoked vasoregression observed in diabetic vasculopathies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (FcεRI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca 2+ level ([Ca 2+ ]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca 2+ signaling in MCs. Fluorescence-based monitoring of [Ca 2+ ]i is a reliable and commonly used approach to study Ca 2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a β-hexosaminidase release assay. The amount of β-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. β-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca 2+ -imaging and degranulation assays.

All three members of the Orai family of cation channels-Orai1, Orai2 and Orai3-are integral membrane proteins that can form store-operated Ca 2+ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca 2+ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca 2+ -dependent exocytosis of inflammatory mediators. We show that the Ca 2+ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca 2+ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2 -/- PMCs at high (2mM) extracellular Ca 2+ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1mM) Ca 2+ concentration. Likewise, the density of CRAC currents, Ca 2+ -dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca 2+ transients, store-operated Ca 2+ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca 2+ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

TRPM4 proteins form Ca(2+)-activated non selective cation (CAN) channels that affect transmembrane Ca(2+)-influx by determining the membrane potential. Tight control of the intracellular Ca(2+) concentration is essential for mast cell responses. In this study, we analyzed the expression of TRPM4 in peritoneal mast cells (PCMC) as a model for connective tissue type mast cells with respect to FcεRI-evoked calcium changes and the subcellular localization of fluorescently labeled TRPM4 using two viral transduction systems before and following antigen stimulation. Our results show that TRPM4 is expressed in PCMCs, is an essential constituent of the endogenous CAN channels in PCMCs and regulates antigen-evoked increases in intracellular calcium that are significantly enhanced in TRPM4-deficient PCMCs. Compared to PCMCs analyzed before antigen stimulation, the cells depict a substantially increased localization of TRPM4 proteins towards the plasma membrane after FcεRI stimulation. Thus, TRPM4 functions as a limiting factor for antigen evoked calcium rise in connective tissue type mast cells and concurrent translocation of TRPM4 into the plasma membrane is part of this mechanism.

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca(2+) influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca(2+) influx. Within the selectivity filter, M(601)-F(609), Y604G strongly reduces avoidance behaviour and eliminates Ca(2+) transients. Y604F also abolishes Ca(2+) transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y(604) may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.

TRPC4 proteins comprise six transmembrane domains, a putative pore-forming region, and an intracellularly located amino- and carboxy-terminus. Among eleven splice variants identified so far, TRPC4α and TRPC4β are the most abundantly expressed and functionally characterized. TRPC4 is expressed in various organs and cell types including the soma and dendrites of numerous types of neurons; the cardiovascular system including endothelial, smooth muscle, and cardiac cells; myometrial and skeletal muscle cells; kidney; and immune cells such as mast cells. Both recombinant and native TRPC4-containing channels differ tremendously in their permeability and other biophysical properties, pharmacological modulation, and mode of activation depending on the cellular environment. They vary from inwardly rectifying store-operated channels with a high Ca(2+) selectivity to non-store-operated channels predominantly carrying Na(+) and activated by Gαq- and/or Gαi-coupled receptors with a complex U-shaped current-voltage relationship. Thus, individual TRPC4-containing channels contribute to agonist-induced Ca(2+) entry directly or indirectly via depolarization and activation of voltage-gated Ca(2+) channels. The differences in channel properties may arise from variations in the composition of the channel complexes, in the specific regulatory pathways in the corresponding cell system, and/or in the expression pattern of interaction partners which comprise other TRPC proteins to form heteromultimeric channels. Additional interaction partners of TRPC4 that can mediate the activity of TRPC4-containing channels include (1) scaffolding proteins (e.g., NHERF) that may mediate interactions with signaling molecules in or in close vicinity to the plasma membrane such as Gα proteins or phospholipase C and with the cytoskeleton, (2) proteins in specific membrane microdomains (e.g., caveolin-1), or (3) proteins on cellular organelles (e.g., Stim1). The diversity of TRPC4-containing channels hampers the development of specific agonists or antagonists, but recently, ML204 was identified as a blocker of both recombinant and endogenous TRPC4-containing channels with an IC50 in the lower micromolar range that lacks activity on most voltage-gated channels and other TRPs except TRPC5 and TRPC3. Lanthanides are specific activators of heterologously expressed TRPC4- and TRPC5-containing channels but can block individual native TRPC4-containing channels. The biological relevance of TRPC4-containing channels was demonstrated by knockdown of TRPC4 expression in numerous native systems including gene expression, cell differentiation and proliferation, formation of myotubes, and axonal regeneration. Studies of TRPC4 single and TRPC compound knockout mice uncovered their role for the regulation of vascular tone, endothelial permeability, gastrointestinal contractility and motility, neurotransmitter release, and social exploratory behavior as well as for excitotoxicity and epileptogenesis. Recently, a single-nucleotide polymorphism (SNP) in the Trpc4 gene was associated with a reduced risk for experience of myocardial infarction.

Transient receptor potential (TRP) proteins form cation channels that are regulated through strikingly diverse mechanisms including multiple cell surface receptors, changes in temperature, in pH and osmolarity, in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), and by phosphoinositides which makes them polymodal sensors for fine tuning of many cellular and systemic processes in the body. The 28 TRP proteins identified in mammals are classified into six subfamilies: TRPC, TRPV, TRPM, TRPA, TRPML, and TRPP. When activated, they contribute to cell depolarization and Ca(2+) entry. In mast cells, the increase of [Ca(2+)](i) is fundamental for their biological activity, and several entry pathways for Ca(2+) and other cations were described including Ca(2+) release activated Ca(2+) (CRAC) channels. Like in other non-excitable cells, TRP channels could directly contribute to Ca(2+) influx via the plasma membrane as constituents of Ca(2+) conducting channel complexes or indirectly by shifting the membrane potential and regulation of the driving force for Ca(2+) entry through independent Ca(2+) entry channels. Here, we summarize the current knowledge about the expression of individual Trp genes with the majority of the 28 members being yet identified in different mast cell models, and we highlight mechanisms how they can regulate mast cell functions. Since specific agonists or blockers are still lacking for most members of the TRP family, studies to unravel their function and activation mode still rely on experiments using genetic approaches and transgenic animals. RNAi approaches suggest a functional role for TRPC1, TRPC5, and TRPM7 in mast cell derived cell lines or primary mast cells, and studies using Trp gene knock-out mice reveal a critical role for TRPM4 in mast cell activation and for mast cell mediated cutaneous anaphylaxis, whereas a direct role of cold- and menthol-activated TRPM8 channels seems to be unlikely for the development of cold urticaria at least in mice.

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

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

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.