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Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

A severe consequence of radiation therapy in patients with head and neck cancer is persistent salivary gland hypofunction which causes xerostomia and oral infections. We previously showed that irradiation (IR) of salivary glands in mice triggers initial transient increases in mitochondrial reactive oxygen species (ROSmt), mitochondrial [Ca2+] ([Ca2+]mt), and activated caspase-3 in acinar cells. In contrast, loss of salivary secretion is persistent. Herein we assessed the role of ROSmt in radiation-induced irreversible loss of salivary gland function. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5 × 3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, ([Ca2+]mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction.

Summary: Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells. : Subedi et al. describe a crucial role for STIM2 in enhancing Orai1 channel function when ER-[Ca2+] is not low enough to stimulate STIM1 activation. Under these conditions, STIM2 recruits STIM1 to ER-PM junctions and triggers a conformational change that causes STIM1 to interact with and induce strong activation of Orai1.

Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Secretion of fluid by salivary glands is stimulated by activation of specific receptors on acinar cell plasma membrane and is mediated by an increase in cytosolic [Ca2+] ([Ca2+]i). The increase in [Ca2+]i regulates a number of ion channels and transporters that are required for establishing an osmotic gradient that drives water flow via aquaporin water channels in the apical membrane. The Store-Operated Ca2+ Entry (SOCE) mechanism, which is regulated in response to depletion of ER-Ca2+, determines the sustained [Ca2+]i increase required for prolonged fluid secretion. Core components of SOCE in salivary gland acinar cells are Orai1 and STIM1. In addition, TRPC1 is a major and non-redundant contributor to SOCE and fluid secretion in salivary gland acinar and ductal cells. Other TRP channels that contribute to salivary flow are TRPC3 and TRPV4, while presence of others, including TRPM8, TRPA1, TRPV1, and TRPV3, have been identified in the gland. Loss of salivary gland function leads to dry mouth conditions, or xerostomia, which is clinically seen in patients who have undergone radiation treatment for head-and-neck cancers, and those with the autoimmune exocrinopathy, Sjögren’s syndrome (pSS). TRPM2 is a unique TRP channel that acts as a sensor for intracellular ROS. We will discuss recent studies reported by us that demonstrate a key role for TRPM2 in radiation-induced salivary gland dysfunction. Further, there is increasing evidence that TRPM2 might be involved in inflammatory processes. These interesting findings point to the possible involvement of TRPM2 in Sjögren’s Syndrome, although further studies will be required to identify the exact role of TRPM2 in this disease.

Store-operated Ca 2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3–mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2 +/+ , but not those from TRPM2 −/− mice, displayed an increase in the concentrations of mitochondrial Ca 2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca 2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers—namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients.

Objective Primary Sjogren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. Methods To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus–mediated gene transfer to the salivary glands of C57BL/6 mice. Results A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector–treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. Conclusion In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6–induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.

Summary Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca2+ influx. The Ca2+-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1−/− mouse SG, agonist-stimulated Ca2+ entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1−/− SG acinar cells, whereas Orai1–STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca2+-activated K+ channel (IK) in the apical region of acinar cell was altered in Cav1−/− SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca2+ influx via TRPC1 channels and was inhibited in Cav1−/− SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1−/− SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1–STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, L-phenylalanine (L-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).

In vivo recycling of nitrate (NO 3 − ) and nitrite (NO 2 − ) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO 3 − is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO 3 − to NO 2 − , which enters circulation and leads to NO generation. The transporters for NO 3 − in salivary glands have not yet been identified. Here we report that sialin ( SLC17A 5 ), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO 3 − /H + cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO 3 − or sialic acid (SA), but not by Br − , and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO 3 − -induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H + -dependent NO 3 − conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO 3 − secretion in saliva after intake of a NO 3 − -rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO 3 − /H + transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.

In clinical islet transplantation, because the long-term insulin-independence rate is still poor, a method for detailed analysis of the transplanted islets in the liver after transplantation is required. We have established a novel imaging technique suitable for analysis of transplanted islets in liver using an optical projection tomography (OPT) method. A three-dimensional tomographic image of the transplanted islets in liver was reconstructed. The number of islets transplanted and the number of transplanted islets observed using OPT showed good correlation. The OPT method was used to compare the numbers of transplanted islets in mouse syngeneic and allogeneic transplantation models. Blood glucose concentrations of streptozotocin (STZ)-induced diabetic mice transplanted with syngeneic islets remained normoglycemic and the number of transplanted islets was largely preserved 11 days after transplantation. In mice transplanted with allogeneic islets, hyperglycemia recurred from 7 days after transplantation and the number and the volume of transplanted islets was significantly reduced 11 days after transplantation. These results indicate that OPT imaging and analysis may be a useful tool to quantitatively and sterically evaluation of transplanted islets in liver at the cellular level.

Islet damage attributed to impaired exocrine cells during pancreas preservation and isolation procedure remains elusive, although released exocrine enzymes could directly damage islets. The aim of this study is to investigate the cellular mechanisms associated with exocrine cells and their possible impact on the islet cell survival and function after isolation. Mouse pancreata were stored in cold University of Wisconsin preservation solution for 0, 24 and 48 h and incubated with or without collagenase at 37°C for 15 min. During preservation, the percentage of exocrine cells with necrosis, which means impaired cellular membrane that allows intracellular enzymes to be released, remains low (10%) regardless of preservation time; whereas the percentage of exocrine cells with apoptosis, which means impaired nucleus and possible intact cellular membrane, increases over time of preservation. After collagenase-free incubation, however, the percentage of exocrine cells with necrosis became higher in longer preservation time, and more than 60% of the necrotic exocrine cells contained apoptosis as well. Islet cells located in pancreata with intact structure are almost kept away either from necrotic or apoptotic changes even after 48 h preservation followed by collagenase-free incubation. However, when islets are isolated after collagenase-containing incubation, the percentage of islet cells with necrosis increases over time of preservation up to approximately 40%. This study suggests that exocrine cells with necrosis could cause damage of isolated islets when the pancreas is dissociated and that the necrosis in exocrine cells might be induced mainly as the conversion from apoptosis that has already existed during preservation.

Store-operated calcium entry (SOCE) regulates critical cellular functions and is determined by precise ER/plasma membrane (PM) junctional interactions. Here we have assessed the effect of hypotonic cell volume increase on SOCE in a salivary gland epithelial cell line (HSG). Thapsigargin (Tg) activated a 2APB- and 1microM Gd(3+)-sensitive, inwardly rectifying, cation current, I(SOC), while hypotonic solution (150mOsm) induced cell swelling and activated an outwardly rectifying cation current that was blocked by 100microM Gd(3+) but not by 2APB. HTS addition before or after Tg attenuated the sensitivity of Ca(2+) influx to 2APB and 1microM Gd(3+). After HTS-induced volume increase, while stimulation of cells with Tg resulted in intracellular Ca(2+) release without Ca(2+) influx, stimulation with CCh caused neither internal Ca(2+) release nor Ca(2+) influx. Importantly, HTS caused the ER to recede from the plasma membrane which prevented Tg-stimulated clustering of STIM1 in the ER/PM region and association of STIM1 with TRPC1 and Orai1. Disruption of SOCE was dependent on the level of hypotonic stress as 225mOsm HTS induced relatively less cell swelling or disruption of SOCE. These results demonstrate that epithelial cells can tolerate small increases (up to 5%) in cell volume while larger increases lead to disruption of ER-PM interactions that are critical for activation of SOCE. We suggest that loss of SOCE could impact cell function and contribute to the deleterious effects of severe hypotonic stress.

Store-operated Ca 2+ entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of ΔERM-STIM1 or STIM1( 684 EE 685 ) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-( 639 KK 640 ) with STIM1( 684 EE 685 ) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1.

Aim Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are major incretins associated with body weight regulation. Dipeptidyl peptidase-IV (DPP-IV) inhibitor increases plasma active GLP-1 and GIP. However, the magnitude of the effects of enhanced GLP-1 and GIP signaling by long-term DPP-IV inhibition on body weight and insulin secretion has not been determined. In this study, we compared the effects of long-term DPP-IV inhibition on body composition and insulin secretion of high fat diet (HFD)-fed wild-type (WT) and GLP-1R knockout (GLP-1R−/−) mice. Main methods HFD-fed WT and GLP-1R−/− mice were treated with or without DPP-IV inhibitor by drinking water. Food and water intake and body weight were measured during 8 weeks of study. CT-based body composition analysis, Oral glucose tolerance test (OGTT), batch incubation study for insulin secretion and quantitative RT-PCR for expression of incretin receptors in isolated islets were performed at the end of study. Key findings DPP-IV inhibitor had no effect on food and water intake and body weight, but increased body fat mass in GLP-1R−/− mice. DPP-IV inhibitor-treated WT and GLP-1R−/− mice both showed increased insulin secretion in OGTT. In isolated islets of DPP-IV inhibitor-treated WT and GLP-1R−/− mice, glucose-induced insulin secretion was increased and insulin secretion in response to GLP-1 or GIP was preserved, without downregulation of incretin receptor expression. Significance Long-term DPP-IV inhibition may maintain body composition through counteracting effects of GLP-1 and GIP while improving glucose tolerance by increasing glucose-induced insulin secretion through the synergistic effects of GLP-1 and GIP.

To clarify the cytoprotective effect of glucagon-like peptide-1 receptor (GLP-1R) signaling in conditions of glucose toxicity in vivo, we performed murine isogenic islet transplantation with and without exendin-4 treatment. When a suboptimal number of islets (150) were transplanted into streptozotocin-induced diabetic mice, exendin-4 treatment contributed to the restoration of normoglycemia. When 50 islets expressing enhanced green fluorescent protein (EGFP) were transplanted, exendin-4 treatment reversed loss of both the number and mass of islet grafts one and 3 days after transplantation. TUNEL staining revealed that exendin-4 treatment reduced the number of apoptotic beta cells during the early posttransplant phase, indicating that GLP-1R signaling exerts its cytoprotective effect on pancreatic beta cells by inhibiting their apoptosis. This beneficial effect might be used both to ameliorate type 2 diabetes and to improve engraftment rates in clinical islet transplantation.

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic β-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3′,5′-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse β-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR ( n = 3–4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion ( n = 8, P < 0.05), and cAMP production ( n = 6, P < 0.01) and insulin secretion ( n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice ( n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.

Agonist-induced Ca 2+ entry via store-operated Ca 2+ (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(−/−) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(−/−) mice. Deletion of TRPC1 also eliminated sustained Ca 2+ -dependent potassium channel activity, which depends on Ca 2+ entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca 2+ signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(−/−) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.

Although store-operated calcium entry (SOCE) was identified more that two decades ago, understanding the molecular mechanisms that regulate and mediate this process continue to pose a major challenge to investigators in this field. Thus, there has been major focus on determining which of the models proposed for this mechanism is valid and conclusively establishing the components of the store-operated calcium (SOC) channel(s). The transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the elusive store-operated Ca(2+) entry channel. While all TRPCs are activated in response to agonist-stimulated phosphatidylinositol 4,5, bisphosphate (PIP(2)) hydrolysis, only some display store-dependent regulation. TRPC1 is currently the strongest candidate component of SOC and is shown to contribute to SOCE in many cell types. Heteromeric interactions of TRPC1 with other TRPCs generate diverse SOC channels. Recent studies have revealed novel components of SOCE, namely the stromal interacting molecule (STIM) and Orai proteins. While STIM1 has been suggested to be the ER-Ca(2+) sensor protein relaying the signal to the plasma membrane for activation of SOCE, Orai1 is reported to be the pore-forming component of CRAC channel that mediates SOCE in T-lymphocytes and other hematopoetic cells. Several studies now demonstrate that TRPC1 also associates with STIM1 suggesting that SOC and CRAC channels are regulated by similar molecular components. Interestingly, TRPC1 is also associated with Orai1 and a TRPC1-Orai1-STIM1 ternary complex contributes to SOC channel function. This review will focus on the diverse SOC channels formed by TRPC1 and the suggestion that TRPC1 might serve as a molecular link that determines their regulation by store-depletion.

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.

In 2012, we reported that 5 out of 11 subjects in a clinical trial (NCT00372320) administering AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first-generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). As subject peak responses were at times much longer (7-42 days) than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. This hypothesis was supported in human salivary gland primary cultures and human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. Conversely, when we transduced mouse and rat cell lines in vitro and submandibular glands in vivo with AdhAQP1, the hCMVp was gradually methylated over time and associated with decreased hAQP1 expression and function in vitro and decreased hAQP1 expression in vivo. These data suggest that the hCMVp in AdhAQP1was probably not methylated in transduced human salivary gland cells of responding subjects, resulting in an unexpectedly longer functional expression of hAQP1.

The authors correct an error in Fig. 1B.

A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists.

Store-operated Ca(2+) entry (SOCE) is activated in response to depletion of intracellular Ca(2+) from the endoplasmic reticulum (ER). A variety of agonists stimulate SOCE via IP(3)-dependent Ca(2+) depletion. SOCE is also activated by thapsigargin, an inhibitor of Ca(2+) reuptake into the ER that induces a net Ca(2+) loss from the ER by unmasking a Ca(2+) "leak" pathway. The molecular identity of this Ca(2+) leak channel and the physiological conditions under which such agonist-independent Ca(2+) depletion might occur remain poorly characterized. In this study, we report that inhibition of the initiation step of protein synthesis (with pactamycin) resulted in detectable Ca(2+) depletion in ER and activation of SOCE. This was completely prevented if the ribosome-nascent chain complexes were first stabilized with an irreversible inhibitor of translational elongation (emetine), suggesting that ER Ca(2+) depletion had occurred through open translocons at the ER. Notably, emetine pretreatment also attenuated thapsigargin-mediated Ca(2+) release and SOCE. Furthermore, both pactamycin and thapsigargin stimulated translocation of STIM1, a protein required for activation of SOCE, to the subplasma membrane region and activated the SOCE-associated current, I (SOC). In aggregate, these data reveal an agonist-independent mechanism for internal Ca(2+) store depletion and activation of SOCE. We suggest that the functional coupling between SOCE and protein synthesis is likely to be critical for maintaining [Ca(2+)](ER) within a range that is required to prevent ER stress during changes in cellular translational activity.

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.

Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4alphaPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.

Mitochondria contribute to cytosolic Ca(2+) homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAG's) induce Ca(2+) release from Ca(2+)-loaded mammalian mitochondria. Release is not mediated by the uni-porter or the Na(+)/Ca(2+) exchanger, nor is it attributed to putative catabolites. DAG's-induced Ca(2+) efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca(2+) and relatively slow reuptake, a secondary progressive release of Ca(2+) occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAG's-induced Ca(2+) efflux is abolished by La(3+) (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca(2+) efflux. Ca(2+)-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAG's-induced Ca(2+) release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAG's-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La(3+). The presence of a second messenger-sensitive Ca(2+) release mechanism in mitochondria could have an important impact on intracellular Ca(2+) homeostasis.

We have reported that internal Ca2+ store depletion in HSY cells stimulates a nonselective cation current which is distinct from ICRAC in RBL cells and TRPC1-dependent ISOC in HSG cells (Liu, X., Groschner, K., and Ambudkar, I. S. (2004) J. Membr. Biol. 200, 93–104). Here we have analyzed the molecular composition of this channel. Both thapsigargin (Tg) and 2-acetyl-sn-glycerol (OAG) stimulated similar non-selective cation currents and Ca2+ entry in HSY cells. The effects of Tg and OAG were not additive. HSY cells endogenously expressed TRPC1, TRPC3, and TRPC4 but not TRPC5 or TRPC6. Immunoprecipitation of TRPC1 pulled down TRPC3 but not TRPC4. Conversely, TRPC1 co-immunoprecipitated with TRPC3. Expression of antisense TRPC1 decreased (i) Tg- and OAG-stimulated currents and Ca2+ entry and (ii) the level of endogenous TRPC1 but not TRPC4. Antisense TRPC3 similarly reduced Ca2+ entry and endogenous TRPC3. Yeast two-hybrid analysis revealed an interaction between NTRPC1 and NTRPC3 (CTRPC1-CTRPC3, CTRPC3-CTRPC1, or CTRPC1-NTRPC3 did not interact), which was confirmed by glutathione S-transferase (GST) pull-down assays (GST-NTRPC3 pulled down TRPC1 and vice versa). Expression of NTRPC1 or NTRPC3 induced similar dominant suppression of Tg- and OAG-stimulated Ca2+ entry. NTRPC3 did not alter surface expression of TRPC1 or TRPC3 but disrupted TRPC1-TRPC3 association. In aggregate, our data demonstrate that TRPC1 and TRPC3 co-assemble, via N-terminal interactions, to form a heteromeric store-operated non-selective cation channel in HSY cells. Thus selective association between TRPCs generate distinct store-operated channels. Diversity of store-operated channels might be related to the physiology of the different cell types.

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of Gαq/11, inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cβ1 and -β2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cβs, Gαq/11, inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion.

Basic fibroblast growth factor (bFGF) and its major receptor FGF receptor-1 (FGFR-1) play an important role in the development of the cortex. The mechanisms underlying the mitogenic role of bFGF/FGFR-1 signaling have not been elucidated. Intracellular Ca2+concentrations ([Ca2+]i) in proliferating cortical neuroepithelial cells are markedly dependent on Ca2+entry (Maric et al., 2000a). The absence of voltage-dependent Ca2+entry channels, which emerge later, indicates that other membrane mechanisms regulate [Ca2+]iduring proliferation. Canonical transient receptor potential (TRPC) family channels are candidates because they are voltage independent and are expressed during CNS development (Strübing et al., 2003).Here, we investigated the involvement of TRPC1 in bFGF-mediated Ca2+entry and proliferation of embryonic rat neural stem cells (NSCs). Both TRPC1 and FGFR-1 are expressed in the embryonic rat telencephalon and coimmunoprecipitate. Quantitative fluorescence-activated cell sorting analyses of phenotyped telencephalic dissociates show that ∼80% of NSCs are TRPC1+, proliferating, and express FGFR-1. Like NSCs profiledex vivo, NSC-derived progeny proliferatingin vitrocoexpress TRPC1 and FGFR1. Antisense knock-down of TRPC1 significantly decreases bFGF-mediated proliferation of NSC progeny, reduces the Ca2+entry component of the Cai2+response to bFGF without affecting Ca2+release from intracellular stores or 1-oleoyl-2-acetyl-sn-glycerol-induced Ca2+entry, and significantly blocks an inward cation current evoked by bFGF in proliferating NSCs. Both Ca2+influx evoked by bFGF and NSC proliferation are attenuated by Gd3+andSKF96365, two antagonists of agonist-stimulated Ca2+entry. Together, these results show that TRPC1 contributes to bFGF/FGFR-1-induced Ca2+influx, which is involved in self-renewal of embryonic rat NSCs.

The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.

TRPC1, a component of store-operated Ca2+ entry (SOCE) channels, is assembled in a complex with caveolin-1 (Cav1) and key Ca2+ signaling proteins. This study examines the role of Cav1 in the function of TRPC1. TRPC1 and Cav1 were colocalized in the plasma membrane region of human submandibular gland and Madin-Darby canine kidney cells. Full-length Cav1 bound to both the N and C termini of TRPC1. Amino acids 271-349, which includes a Cav1 binding motif (amino acids 322-349), was identified as the Cav1 binding domain in the TRPC1 N terminus. Deletion of amino acids 271-349 or 322-349 prevented plasma membrane localization of TRPC1. Importantly, TRPC1Delta271-349 induced a dominant suppression of SOCE and was associated with wild-type TRPC1. Although the role of the C-terminal Cav1 binding domain is not known, its deletion did not affect localization of TRPC1 (Singh, B. B., Liu, X., and Ambudkar, I. S. (2000) J. Biol. Chem. 275, 36483-36486). Further, expression of a truncated Cav1 (Cav1Delta51-169), but not full-length Cav1, similarly disrupted plasma membrane localization of endogenously and exogenously expressed TRPC1 in human submandibular gland and Madin-Darby canine kidney cells. Cav1Delta51-169 also suppressed thapsigarginand carbachol-stimulated Ca2+ influx and increased the detergent solubility of TRPC1, although plasma membrane lipid raft domains were not disrupted. These data demonstrate that plasma membrane localization of TRPC1 depends on an interaction between its N terminus and Cav1. Thus, our data suggest that Cav1 has an important role in the assembly of SOCE channel(s).

The exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557–620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, ISOC, were not altered. Expression of TRPC1Δ567–793, but not TRPC1Δ664–793, induced a similar decrease in SOCC activity. Furthermore, TRPC1Δ567–793 was co-immunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6 region (Asp → Asn and Glu → Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left shift in E rev. Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx.

Aquaporins are a family of water channels considered to play an important role in fluid transport across plasma membranes. Among the reported isoforms, relatively little is known about the functional role of aquaporin 8 (AQP8), and there are no cell lines known to express the AQP8 protein. We report here that the rat submandibular epithelial cell line, SMIE, expresses AQP8. Using RT-PCR, the presence of mRNA for AQP8 was demonstrated in these cells. Confocal immunofluorescence experiments revealed that the AQP8 protein is primarily present in the apical membranes of SMIE cells. When grown as a polarized monolayer on collagen coated polycarbonate filters, and exposed on their apical surface to different hyperosmotic (440, 540, or 640 mOsm) solutions, net fluid movement across SMIE cells was 8-25-fold that seen under isosmotic conditions. Similarly, when grown on coverslips and then exposed to a hypertonic solution, SMIE cells shrunk as a function of time. Together, these results suggest that SMIE cells endogenously express functional AQP8 water channels.