A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis.

Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.

Presynaptic α2δ-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring.

Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Post-mortem histology in transient receptor potential cation channel subfamily V member 6 (TRPV6) under-mineralising skeletal dysplasia suggests postnatal skeletal recovery: a case report.

BACKGROUND: The calcium-selective channel TRPV6 (transient receptor potential cation channel subfamily V member 6) is crucial for maternal-fetal calcium transport across the placenta. TRPV6 mutations have recently been associated with an antenatally severe under-mineralising skeletal dysplasia accompanied by postnatal biochemical abnormalities. This is the first post-mortem report in a patient with TRPV6 skeletal dysplasia. CASE PRESENTATION: The female infant had severe antenatal and postnatal skeletal abnormalities by 20 weeks gestation and was ventilator-dependent from birth. These skeletal abnormalities were apparent at an earlier gestational age than in previous reported cases and a more severe clinical course ensued. Biochemical and skeletal abnormalities, including bone density, improved postnatally but cardiac arrest at 4 months of age led to withdrawal of intensive care. Compound heterozygous TRPV6 variants (c.1978G > C p.(Gly660Arg) and c.1528C > T p.(Arg510Ter)) were identified on exome sequencing. Post-mortem identified skeletal abnormalities but no specific abnormalities in other organ systems. No placental pathology was found, multi-organ histological features reflected prolonged intensive care only. Post-mortem macroscopic examination indicated reduced thoracic size and short, pale and pliable ribs. Histological examination identified reduced number of trabeculae in the diaphyses (away from the growth plates), whereas metaphyses showed adequate mineralisation and normal number of trabeculae, but with slightly enlarged reactive chondrocytes, indicating post-natal skeletal growth recovery. Post-mortem radiological findings demonstrated improved bone density, improved rib width, healed fractures, although ribs were still shorter than normal. Long bones (especially humerus and femur) had improved from initial poorly defined metaphyses and reduced bone density to sharply defined metaphyses, prominent growth restart lines in distal diaphyses and bone-in-bone appearance along diaphyses. CONCLUSIONS: This case provide bone histological confirmation that human skeletal development is compromised in the presence of TRPV6 pathogenic variants. Post-mortem findings were consistent with abnormal in utero skeletal mineralisation due to severe calcium deficit from compromised placental calcium transfer, followed by subsequent phenotypic improvement with adequate postnatal calcium availability. Significant skeletal recovery occurs in the early weeks of postnatal life in TRPV6 skeletal dysplasia.

Overexpression of Osmosensitive Ca2+-Permeable Channel TMEM63B Promotes Migration in HEK293T Cells.

The recent discovery of the osmosensitive calcium (Ca2+) channel OSCA has revealed the potential mechanism by which plant cells sense diverse stimuli. Osmosensory transporters and mechanosensitive channels can detect and respond to osmotic shifts that play an important role in active cell homeostasis. Members of the TMEM63 family of proteins are described as the closest homologues of OSCAs. Here, we characterize TMEM63B, a mammalian homologue of OSCAs, recently classified as mechanosensitive. In HEK293T cells, TMEM63B localizes to the plasma membrane and is associated with F-actin. This Ca2+-permeable channel specifically induces Ca2+ influx across the membrane in response to extracellular Ca2+ concentration and hyperosmolarity. In addition, overexpression of TMEM63B in HEK293T cells significantly enhanced cell migration and wound healing. The link between Ca2+ osmosensitivity and cell migration might help to establish TMEM63B's pathogenesis, for example, in cancer in which it is frequently overexpressed.

SMDT1-driven change in mitochondrial dynamics mediate cell apoptosis in PDAC.

Mitochondrial Ca2+ uptake, an important governing for Ca2+ homeostasis, is catalyzed by the mitochondrial calcium uniporter (MCU) complex. SMDT1, as a subunit of MCU complex, was essential for bridging the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU. However, the molecular mechanism and regulatory purpose of SMDT1 remain largely unexplored, especially no study was reported in cancer. Here, we firstly reported that how SMDT1 exerted its role through mediating mitochondrial dynamic in PDAC malignancy. In this study, by screening online of subunit of MCU complex, we confirmed that SMDT1 expression was significantly positive correlated with PDAC prognosis. The GEO datasets showed decreased SMDT1 expression in PDAC tumor compared with non-tumor tissues. SMDT1 overexpression could notably inhibit cell proliferation and induce cell apoptosis. Further analysis demonstrated that up-regulated SMDT1 in ASPC1 and Canpan1 cells led to increased accumulation of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx. And more releasing of cytochrome c located in cytosolic. Mechanistically, in the morphological analysis of mitochondria, more fragmented mitochondria were presented in SMDT1 overexpression cells by promting the phosphorylation of Drp1, increasing Fis and decreasing MFN1. Meanwhile, more Drp1 was translocated on the mitochondrial from the cytoplasm in up-regulated SMDT1 cells. On the basis of the evidence above we deduce that SMDT1-driven change in mitochondrial dynamics mediated cells apoptosis in PDAC. And, SMDT1 could serve as an important therapeutic target to normalize mitochondrial dynamic responsible for poor prognosis in PDAC.

TPC2 controls pigmentation by regulating melanosome pH and size.

Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

Calcium channels in the development, maturation, and function of spermatozoa.

A proper dialogue between spermatozoa and the egg is essential for conception of a new individual in sexually reproducing animals. Ca(2+) is crucial in orchestrating this unique event leading to a new life. No wonder that nature has devised different Ca(2+)-permeable channels and located them at distinct sites in spermatozoa so that they can help fertilize the egg. New tools to study sperm ionic currents, and image intracellular Ca(2+) with better spatial and temporal resolution even in swimming spermatozoa, are revealing how sperm ion channels participate in fertilization. This review critically examines the involvement of Ca(2+) channels in multiple signaling processes needed for spermatozoa to mature, travel towards the egg, and fertilize it. Remarkably, these tiny specialized cells can express exclusive channels like CatSper for Ca(2+) and SLO3 for K(+), which are attractive targets for contraception and for the discovery of novel signaling complexes. Learning more about fertilization is a matter of capital importance; societies face growing pressure to counteract rising male infertility rates, provide safe male gamete-based contraceptives, and preserve biodiversity through improved captive breeding and assisted conception initiatives.

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis.

TRPA1 receptor is upregulated in human oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology with antigen-specific and non-specific mechanisms. Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel activated by noxious stimuli such as oxidative stress products evoking pain and release of proinflammatory mediators from sensory nerve endings culminating in neurogenic inflammation. Extraneuronal TRPA1s, for example, on immune cells possess yet unknown functions. SUBJECTS AND METHODS: We studied the buccal mRNA expression (qPCR) and protein localization (immunohistochemistry) of TRPA1 receptors and key OLP mediator transcripts in oral mucosa samples of healthy volunteers (n = 9), OLP patients (n = 43), and OLP-like hyperkeratotic patients (n = 12). RESULTS: We measured 27.7- and 25.5-fold TRPA1 mRNA increase in OLP and OLP-like hyperkeratotic patients compared to healthy controls. TRPA1 transcripts elevated 2.4-fold in hypertensive OLP but not in hyperkeratotic patients compared to counterparts, reduced by 1.6-fold by angiotensin-convertase inhibitor intake. TRPA1 messenger RNA was more coexpressed with transcripts of tumor necrosis factor α than with interferon γ. Keratinocytes, macrophages but not T cells expressed TRPA1. CONCLUSIONS: We provided evidence for the extraneuronal presence and upregulation of the proinflammatory TRPA1 receptor in buccal samples of patients with OLP. This may implicate the ion channel in the pathomechanism of OLP.

Inhibitors of Calcium-Activated Anion Channels Modulate Hypnotic Ethanol Responses in Adult Sprague Dawley Rats.

BACKGROUND: Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. METHODS: We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. RESULTS: Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. CONCLUSIONS: These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane.

BACKGROUND: Orai/CRACM1 ion channels provide the major Ca(2+) influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca(2+)-activated K(+) channel KCa3.1 modulates Ca(2+) influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca(2+)- and K(+)-selective channels. RESULTS: Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca(2+), and attenuated in the presence of the selective Orai blocker GSK-7975A. Currents elicited by the KCa3.1 opener 1-EBIO were also attenuated by GSK-7975A. The Orai1 E106Q dominant-negative mutant ablated 1-EBIO and FcεRI-dependent KCa3.1 currents in HLMCs. Orai1 but not Orai2 was shown to co-immunoprecipitate with KCa3.1 when overexpressed in HEK293 cells, and Orai1 and KCa3.1 were seen to co-localise in the HEK293 plasma membrane using confocal microscopy. CONCLUSION: KCa3.1 activation in HLMCs is highly dependent on Ca(2+) influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels.

Increased expression of three types of transient receptor potential channels (TRPA1, TRPV4 and TRPV3) in burn scars with post-burn pruritus.

Post-burn pruritus is a common distressing consequence of burn wounds. Empirical treatment often fails to have a satisfactory outcome on post-burn pruritus, as the mechanism of post-burn pruritus has not been fully elucidated. The aim of this study was to evaluate the manifestation of transient receptor potential (TRP) channels in post-burn pruritus. Fifty-one burn patients with (n=33) or without (n=18) pruritus were investigated, including skin biopsies. Not unexpectedly, the scarred body area was larger in the former group. In immunohistochemistry, TPRV3 was significantly elevated in the epidermis of burn scars with pruritus. Furthermore, real time- PCR showed that mRNA of TRPA1 and TRPV4 was increased in itching burn scars. Staining for substance P and CGRP did not differ between the 2 grouped, but the former neuropeptide was increased in burn scars. These results may help determine a specific therapeutic approach for post-burn pruritus.

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

Store-operated Ca²+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I(SOC), activated in response to Ca²+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I(CRAC); the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³+, removal of extracellular Ca²+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²+-containing, but not Ca²+-free, medium. Consistent with this, I(CRAC) is activated in cells pretreated with thapsigargin in Ca²+-free medium while I(SOC) is activated in cells pretreated in Ca²+-containing medium. Significantly, TRPC1 function is required for sustained K(Ca) activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

Down-regulation of Stargazin inhibits the enhanced surface delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor GluR1 subunit in rat dorsal horn and ameliorates postoperative pain.

BACKGROUND: Stargazin is the first transmembrane protein known to regulate synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. However, it is unclear whether regulation of the surface delivery of spinal AMPA receptor subunits by stargazin contributes to postoperative pain development. METHODS: Western blot analysis was used to examine changes in the surface delivery of AMPA receptor subunits, GluR1 and GluR2, in rat dorsal horn. The interaction between stargazin and GluR1 and GluR2 was examined by coimmunoprecipitation. Expression of stargazin was suppressed by intrathecal administration of small interfering RNA311. RESULTS: Membrane-bound GluR1, but not GluR2, in ipsilateral dorsal horn was increased at 3 h (1.49 ± 0.15-fold of ß-tubulin, mean ± SEM) and 1 day (1.03 ± 0.25) after incision, as compared with that in control rats (naive, 0.63 ± 0.23, P < 0.05, n = 6 per group). The amount of GluR1 coimmunoprecipitated with stargazin was greater at 3 h after incision (1.48 ± 0.31-fold of input) than that in control animals (0.45 ± 0.24, P < 0.05, n = 6 per group). Importantly, the increase in membrane GluR1 at 3 h after incision was normalized to near control level (0.72 ± 0.20-fold of ß-tubulin) by pretreatment with intrathecal stargazin small interfering RNA311 (0.87 ± 0.09), but not scrambled small interfering RNA (1.48 ± 0.24) or vehicle (1.25 ± 0.13, P < 0.05, n = 6 per group). Stargazin small interfering RNA311 pretreatment prevented the increase in stargazin-GluR1 interaction and decreased postoperative pain after incision. CONCLUSIONS: This study suggests a critical role of stargazin-mediated surface delivery of GluR1 subunit in the development of postoperative pain. A better therapeutic strategy for postoperative pain may involve selectively down-regulating spinal stargazin to inhibit synaptic targeting of GluR1 subunit.

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells.

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Food allergen--induced mast cell degranulation is dependent on PI3K-mediated reactive oxygen species production and upregulation of store-operated calcium channel subunits.

The importance of Ca(2+) influx via store-operated calcium channels (SOCs) leading to mast cell degranulation is well known in allergic disease. However, the underlying mechanisms are not fully understood. With food-allergic rat model, the morphology of degranulated mast cell was analysed by toluidine blue stain and electron microscope. Ca(2+) influx via SOCs was checked by Ca(2+) imaging confocal microscope. Furthermore, the mRNA and protein expression of SOCs subunits were investigated using qPCR and Western blot. We found that ovalbumin (OVA) challenge significantly increased the levels of Th2 cytokines and OVA-specific IgE in allergic animals. Parallel to mast cell activation, the levels of histamine in serum and supernatant of rat peritoneal lavage solution were remarkably increased after OVA treatment. Moreover, the Ca(2+) entry through SOCs evoked by thapsigargin was increased in OVA-challenged group. The mRNA and protein expressions of SOC subunits, stromal interaction molecule 1 (STIM1) and Orail (calcium-release-activated calcium channel protein 1), were dramatically elevated under food-allergic condition. Administration of Ebselen, a scavenger of reactive oxygen species (ROS), significantly attenuated OVA sensitization-induced intracellular Ca(2+) rise and upregulation of SOCs subunit expressions. Intriguingly, pretreatment with PI3K-specific inhibitor (Wortmannin) partially abolished the production of ROS and subsequent elevation of SOCs activity and their subunit expressions. Taken together, these results imply that enhancement of SOC-mediated Ca(2+) influx induces mast cell activation, contributing to the pathogenesis of OVA-stimulated food allergy. PI3K-dependent ROS generation involves in modulating the activity of SOCs by increasing the expressions of their subunit.

Store-operated Ca2+ entry is involved in transforming growth factor-ß1 facilitated proliferation of rat airway smooth muscle cells.

OBJECTIVE: To investigate the role and underlying mechanisms of store-operated Ca(2+) entry (SOCE) in mediating the promoting effect of transforming growth factor (TGF)-ß1 on the proliferation of airway smooth muscle cells (ASMCs). METHODS: Rat bronchial smooth muscle cells were cultured as we described previously. The intracellular Ca(2+) concentration ([Ca(2+)]i) of ASMCs was measured by laser confocal microscope Ca(2+) fluorescence imaging with Fluo-3/AM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and p27 expression assay were used to determine the proliferation rate of ASMCs. RESULTS: We demonstrated that TGF-ß1 (10 ng/ml) increased basal (Ca(2+)]i) level, [Ca(2+)]i rise induced by thapsigargin-induced Ca(2+) release and SOCE in rat ASMCs. This effect of TGF-ß1 on SOCE was not inhibited by glucocorticoid dexamethasone (DXM, 100 nM), antioxidant α-tocopherol (100 µM), and intermediate-conductance Ca(2+)-activated K(+) channels (IKCa) inhibitor charybdotoxin (100 nM), suggesting that reactive oxygen species and IKCa channels might not mediate the effect of TGF-ß1. TGF-ß1 slightly increased the expression of Orai1 and STIM1, two important molecules involved in the molecule component and regulation of SOC channels, in the presence of 10% fetal bovine serum (FBS). The proliferation of ASMC stimulated with 2.5% FBS was promoted by TGF-ß1, and partly inhibited by non-specific Ca(2+) channel blocker SKF-96365 (10 µM) and Ni(2+) (100 µM). DXM, α-tocopherol, and charybdotoxin had no effect on the proliferation promoted by TGF-ß1. CONCLUSION: TGF-ß1 promotes ASMC proliferation partly through increasing the expression and activity of SOC channels.

The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit.

Stargazer mice have spike-wave seizures characteristic of absence epilepsy, with accompanying defects in the cerebellum and inner ear. We describe here a novel gene, Cacng2, whose expression is disrupted in two stargazer alleles. It encodes a 36-kD protein (stargazin) with structural similarity to the gamma subunit of skeletal muscle voltage-gated calcium (Ca2+) channels. Stargazin is brain-specific and, like other neuronal Ca2+-channel subunits, is enriched in synaptic plasma membranes. In vitro, stargazin increases steady-state inactivation of alpha1 class A Ca2+ channels. The anticipated effect in stargazer mutants, inappropriate Ca2+ entry, may contribute to their more pronounced seizure phenotype compared with other mouse absence models with Ca2+-channel defects. The discovery that the stargazer gene encodes a gamma subunit completes the identification of the major subunit types for neuronal Ca2+ channels, namely alpha1, alpha2delta, beta and gamma, providing a new opportunity to understand how these channels function in the mammalian brain and how they may be targeted in the treatment of neuroexcitability disorders.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

Plasmalemmal caveolae and GPI-anchored membrane proteins.

Caveolae are essential endocytic organelles that use glycosyl-phosphatidyl-inositol (GPI)-anchored membrane proteins to concentrate low molecular weight substances before delivery to the cell. Caveolae and GPI-anchored proteins are uniquely adapted for this task. Recent advances suggest that this endocytic pathway also has an important role in modulating the interaction of a cell with its environment. Many vital functions that occur in this organelle remain to be discovered.