RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity.

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

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.

STIM-TRP Pathways and Microdomain Organization: Contribution of TRPC1 in Store-Operated Ca2+ Entry: Impact on Ca2+ Signaling and Cell Function.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ entry pathway that is activated in response to depletion of ER-Ca2+ stores and critically controls the regulation of physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), which are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. While TRPC1 was associated with SOCE and regulation of function in several cell types, none of the TRPC members displayed I CRAC, the store-operated current identified in lymphocytes and mast cells. Intensive search finally led to the identification of Orai1 and STIM1 as the primary components of the CRAC channel. Orai1 was established as the pore-forming channel protein and STIM1 as the ER-Ca2+ sensor protein involved in activation of Orai1. STIM1 also activates TRPC1 via a distinct domain in its C-terminus. However, TRPC1 function depends on Orai1-mediated Ca2+ entry, which triggers recruitment of TRPC1 into the plasma membrane where it is activated by STIM1. TRPC1 and Orai1 form distinct store-operated Ca2+ channels that regulate specific cellular functions. It is now clearly established that regulation of TRPC1 trafficking can change plasma membrane levels of the channel, the phenotype of the store-operated Ca2+ current, as well as pattern of SOCE-mediated [Ca2+]i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca2+]i signal generated by Orai1. This review will highlight current concepts of the activation and regulation of TRPC1 channels and its impact on cell function.

Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.

Calcium signalling in salivary gland physiology and dysfunction.

Studies over the past four decades have established that Ca(2+) is a critical factor in control of salivary gland function and have led to identification of the critical components of this process. The major ion transport mechanisms and ion channels that are involved in fluid secretion have also been established. The key event in activation of fluid secretion is an increase in [Ca(2+) ]i triggered by inositol 1,4,5-trisphosphate (IP3 )-induced release of Ca(2+) from ER via the IP3 receptor (IP3 R). IP3 Rs determine the site of initiation and the pattern of the [Ca(2+) ]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+) ]i and fluid secretion and is mediated by the store-operated Ca(2+) entry (SOCE) mechanism. Orai1, TRPC1, TRPC3 and STIM1 have been identified as critical components of SOCE in these cells. Cells finely tune the generation and amplification of [Ca(2+) ]i signals for regulation of cell function. An important emerging area is the concept that unregulated [Ca(2+) ]i signals in cells can directly cause cell damage, dysfunction and disease. Alternatively, aberrant [Ca(2+) ]i signals can also amplify and increase the rates of cell damage. Such defects in Ca(2+) signalling have been described in salivary glands in conjunction with radiation-induced loss of salivary gland function as well as in the salivary defects associated with the autoimmune exocrinopathy Sjögren's syndrome. Such defects have been associated with altered function or expression of key Ca(2+) signalling components, such as STIM proteins and TRP channels. These studies offer new avenues for examining the mechanisms underlying the disease and development of novel clinical targets and therapeutic strategies.

Fast endocytic recycling determines TRPC1-STIM1 clustering in ER-PM junctions and plasma membrane function of the channel.

Stromal interaction molecule 1 (STIM1) senses depletion of ER-Ca2+ store and clusters in ER-PM junctions where it associates with and gates Ca2+ influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2+ entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER-PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1-STIM1 clustering within ER-PM junctions following ER-Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells.

Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

Role of TRPC Channels in Store-Operated Calcium Entry.

Store-operated calcium entry (SOCE) is a ubiquitous Ca(2+) entry pathway that is activated in response to depletion of Ca(2+) stores within the endoplasmic reticulum (ER) and contributes to the control of various physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), that are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. TRPC channels show a miscellany of tissue expression, physiological functions and channel properties. However, none of the TRPC members display currents that resemble I CRAC. Intensive search for the CRAC channel component led to identification of Orai1 and STIM1, now established as being the primary constituents of the CRAC channel. There is now considerable evidence that STIM1 activates both Orai1 and TRPC1 via distinct domains in its C-terminus. Intriguingly, TRPC1 function is not only dependent on STIM1 but also requires Orai1. The critical functional interaction between TRPC1 and Orai1, which determines the activation of TRPC1, has also been identified. In this review, we will discuss current concepts regarding the role of TRPC channels in SOCE, the physiological functions regulated by TRPC-mediated SOCE, and the complex mechanisms underlying the regulation of TRPCs, including the functional interactions with Orai1 and STIM1.

Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1.

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-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 Ca(2+) 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 Ca(2+)-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 Ca(2+) 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.

TRPC1 contributes to the Ca2+-dependent regulation of adenylate cyclases.

SOCE (store-operated Ca2+ entry) is mediated via specific plasma membrane channels in response to ER (endoplasmic reticulum) Ca2+ store depletion. This route of Ca2+ entry is central to the dynamic interplay between Ca2+ and cAMP signalling in regulating the activity of Ca2+-sensitive adenylate cyclase isoforms (AC1, AC5, AC6 and AC8). Two proteins have been identified as key components of SOCE: STIM1 (stromal interaction molecule 1), which senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion, where it then activates Orai1, the pore-forming component of the CRAC (Ca2+ release-activated Ca2+) channel. Previous studies reported that co-expression of STIM1 and Orai1 in HEK-293 (human embryonic kidney 293) cells enhances Ca2+-stimulated AC8 activity and that AC8 and Orai1 directly interact to enhance this regulation. Nonetheless, the additional involvement of TRPC (transient receptor potential canonical) channels in SOCE has also been proposed. In the present study, we evaluate the contribution of TRPC1 to SOCE-mediated regulation of Ca2+-sensitive ACs in HEK-293 cells stably expressing AC8 (HEK-AC8) and HSG (human submandibular gland) cells expressing an endogenous Ca2+-inhibited AC6. We demonstrate a role for TRPC1 as an integral component of SOCE, alongside STIM1 and Orai1, in regulating Ca2+ fluxes within AC microdomains and influencing cAMP production.

STIM1 and STIM2 protein deficiency in T lymphocytes underlies development of the exocrine gland autoimmune disease, Sjogren's syndrome.

Primary Sjögren's Syndrome (pSS) is an autoimmune disease involving salivary and other exocrine glands that leads to progressive lymphocytic infiltration into the gland, tissue damage, and secretory defects. The mechanism underlying this disease remains poorly understood. Here we report that mice with T-cell-targeted deletion of Stromal Interaction Molecule (STIM) 1 and STIM2 [double-knockout (DKO)] mice develop spontaneous and severe pSS-like autoimmune disease, displaying major hallmarks of the disease. In DKO mice, diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by age 6 wk, progressing to severe inflammation by age 12 wk. Sjögren's syndrome-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, and progressive salivary gland destruction and loss of fluid secretion were also seen. Importantly, we report that peripheral blood mononuclear cells as well as lymphocytic infiltrates in submandibular glands from patients with pSS demonstrated significant reductions in STIM1 and STIM2 proteins. Store-operated calcium entry was also reduced in peripheral blood mononuclear cells from pSS patients compared with those from healthy controls. Thus, deficiency of STIM1 and STIM2 proteins in T cells, and consequent defects in Ca(2+) signaling, are associated with salivary gland autoimmunopathy in DKO mice and pSS patients. These data reveal a previously unreported link between STIM1 and STIM2 proteins and pSS.

Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane.

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 (SLC17A5), 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.

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.

Physiological functions and regulation of TRPC channels.

The TRP-canonical (TRPC) subfamily, which consists of seven members (TRPC1-TRPC7), are Ca(2+)-permeable cation channels that are activated in response to receptor-mediated PIP2 hydrolysis via store-dependent and store-independent mechanisms. These channels are involved in a variety of physiological functions in different cell types and tissues. Of these, TRPC6 has been linked to a channelopathy resulting in human disease. Two key players of the store-dependent regulatory pathway, STIM1 and Orai1, interact with some TRPC channels to gate and regulate channel activity. The Ca(2+) influx mediated by TRPC channels generates distinct intracellular Ca(2+) signals that regulate downstream signaling events and consequent cell functions. This requires localization of TRPC channels in specific plasma membrane microdomains and precise regulation of channel function which is coordinated by various scaffolding, trafficking, and regulatory proteins.

Polarized but differential localization and recruitment of STIM1, Orai1 and TRPC channels in secretory cells.

Polarized Ca(2+) signals in secretory epithelial cells are determined by compartmentalized localization of Ca(2+) signaling proteins at the apical pole. Recently the ER Ca(2+) sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store-dependent Ca(2+) influx. STIM1 also gates the transient receptor potential-canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca(2+) influx and frequency of Ca(2+) oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP(3) receptors (IP(3) Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2-aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca(2+) entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca(2+) permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca(2+) ] oscillations and thus impact critical cellular functions.

Extracellular Ca(2+) sensing in salivary ductal cells.

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).

Contribution and regulation of TRPC channels in store-operated Ca2+ entry.

Store-operated calcium entry (SOCE) is activated in response to depletion of the endoplasmic reticulum-Ca(2+) stores following stimulation of plasma membrane receptors that couple to PIP2 hydrolysis and IP3 generation. Search for the molecular components of SOCE channels led to the identification of mammalian transient receptor potential canonical (TRPC) family of calcium-permeable channels (TRPC1-TRPC7), which are all activated in response to stimuli that result in PIP2 hydrolysis. While several TRPCs, including TRPC1, TRPC3, and TRPC4, have been implicated in SOCE, the data are most consistent for TRPC1. Extensive studies in cell lines and knockout mouse models have established the contribution of TRPC1 to SOCE. Furthermore, there is a critical functional interaction between TRPC1 and the key components of SOCE, STIM1, and Orai1, which determines the activation of TRPC1. Orai1-mediated Ca(2+) entry is required for recruitment of TRPC1 and its insertion into surface membranes while STIM1 gates the channel. Notably, TRPC1 and Orai1 generate distinct patterns of Ca(2+) signals in cells that are decoded for the regulation of specific cellular functions. Thus, SOCE appears to be a complex process that depends on temporal and spatial coordination of several distinct steps mediated by proteins in different cellular compartments. Emerging data suggest that, in many cell types, the net Ca(2+) entry measured in response to store depletion is the result of the coordinated regulation of different calcium-permeable ion channels. Orai1 and STIM1 are central players in this process, and by mediating recruitment or activation of other Ca(2+) channels, Orai1-CRAC function can elicit rapid changes in global and local [Ca(2+)]i signals in cells. It is most likely that the type of channels and the [Ca(2+)]i signature that are generated by this process reflect the physiological function of the cell that is regulated by Ca(2+).

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets.

Tumor vascularization is a critical process that determines tumor growth and metastasis. In the last decade new experimental evidence obtained from in vitro and in vivo studies have challenged the classical angiogenesis model forcing us to consider new scenarios for tumor neovascularization. In particular, the genetic stability of tumor-derived endothelial cells (TECs) has been recently questioned in several studies, which show that TECs, as well as pericytes, differ significantly from their normal counterparts at genetic and functional levels. In addition to such an epigenetic action of tumor microenvironment on endothelial cells (ECs) commitment, the distinct characteristics of TECs could be due to differences in their origin compared with preexisting differentiated ECs. Intracellular Ca(2+) signals are involved at different critical phases in the regulation of the complex process of angiogenesis and tumor progression. These signals are generated by a wide variety of intrinsic and extrinsic factors. Several key components of Ca(2+) signaling including Ca(2+) channels in the plasma membrane, endoplasmic reticulum, calcium pumps, and mitochondria contribute to the generation, amplitude, and frequency of these Ca(2+) change. In particular, several members of the transient receptor potential (TRP) family of calcium-permeable channels have profound effects on the function of ECs. Because of its multifaceted role in the control of cell function, proliferation, and motility, TRP channels have been suggested as a potential molecular target for control of tumor neovascularization. Since plasma membrane Ca(2+) channels are easily and directly accessible via the bloodstream, they are potential targets for a number of pharmacological and antibody-targeted therapeutic strategies, with specificity being the main limitation. In this review we discuss recent advances in understanding the role of Ca(2+) channels, with specific reference to TRP channels, in tumor vascularization process.

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1.

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 DeltaERM-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.