Biswaranjan Pani

Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components.

Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca2+ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca2+-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and ISOC in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of ISOC in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca2+ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.

Lipid rafts/caveolae as microdomains of calcium signaling

Ca(2+) is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca(2+) that is achieved by the activation of several plasma membrane (PM) Ca(2+) channels as well as release of Ca(2+) from the internal stores. One such channel is referred to as store-operated Ca(2+) channel that is activated by the release of endoplasmic reticulum (ER) Ca(2+) which initiates store-operated Ca(2+) entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca(2+) signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca(2+) signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca(2+) channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca(2+)-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca(2+)-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.

Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice.

Agonist-induced Ca2+ entry via store-operated Ca2+ (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 Ca2+-dependent potassium channel activity, which depends on Ca2+ entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca2+ 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.

Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca2+ entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca2+ store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1D76A, which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1D76A puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.

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

Store-operated Ca2+ 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(684EE685) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-(639KK640) with STIM1(684EE685) 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.

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

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.

Helminth Induced Suppression of Macrophage Activation Is Correlated with Inhibition of Calcium Channel Activity

Helminth parasites cause persistent infections in humans and yet many infected individuals are asymptomatic. Neurocysticercosis (NCC), a disease of the central nervous system (CNS) caused by the cestode Taenia solium, has a long asymptomatic phase correlated with an absence of brain inflammation. However, the mechanisms of immune suppression remain poorly understood. Here we report that murine NCC displays a lack of cell surface maturation markers in infiltrating myeloid cells. Furthermore, soluble parasite ligands (PL) failed to induce maturation of macrophages, and inhibited TLR-induced inflammatory cytokine production. Importantly, PL treatment abolished both LPS and thapsigargin-induced store operated Ca2+ entry (SOCE). Moreover, electrophysiological recordings demonstrated PL-mediated inhibition of LPS or Tg-induced currents that were TRPC1-dependent. Concomitantly STIM1-TRPC1 complex was also impaired that was essential for SOCE and sustained Ca2+ entry. Likewise loss of SOCE due to PL further inhibited NFkB activation. Overall, our results indicate that the negative regulation of agonist induced Ca2+ signaling pathway by parasite ligands may be a novel immune suppressive mechanism to block the initiation of the inflammatory response associated with helminth infections.

Effect of cell swelling on ER/PM junctional interactions and channel assembly involved in SOCE.

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.

Neurological and Motor Disorders: Neuronal Store-Operated Ca 2+ Signaling: An Overview and Its Function

Calcium (Ca2+) is a ubiquitous second messenger that performs significant physiological task such as neurosecretion, exocytosis, neuronal growth/differentiation, and the development and/or maintenance of neural circuits. An important regulatory aspect of neuronal Ca2+ homeostasis is store-operated Ca2+ entry (SOCE) which, in recent years, has gained much attention for influencing a variety of nerve cell responses. Essentially, activation of SOCE ensues following the activation of the plasma membrane (PM) store-operated Ca2+ channels (SOCC) triggered by the depletion of endoplasmic reticulum (ER) Ca2+ stores. In addition to the TRPC (transient receptor potential canonical) and the Orai family of ion channels, STIM (stromal interacting molecule) proteins have been baptized as key molecular regulators of SOCE. Functional significance of the TRPC channels in neurons has been elaborately studied; however, information on Orai and STIM components of SOCE, although seems imminent, is currently limited. Importantly, perturbations in SOCE have been implicated in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of SOCC in neurodegeneration would presumably unveil avenues for plausible therapeutic interventions. We thus review the role of SOCE-regulated neuronal Ca2+ signaling in selecting neurodegenerative conditions.