The volume-regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling.

The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c-/- mice have increased cell cycle progression, proliferation, survival, Ca2+ influx and cytokine production-a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c-/- mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING-p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity.

Functional determination of calcium-binding sites required for the activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors, including IP3, Ca2+, and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring regulation by Ca2+ have yet to be ascertained. Using comparative structural analysis of Ca2+-binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single-channel open probability (P0), and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues is neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+-binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well-conserved activation site is a common underlying mechanism resulting in increased channel activity shared by IP3Rs and RyRs.

Sex-Specific Platelet Activation Through Protease-Activated Receptors Reverses in Myocardial Infarction.

OBJECTIVE: The platelet phenotype in certain patients and clinical contexts may differ from healthy conditions. We evaluated platelet activation through specific receptors in healthy men and women, comparing this to patients presenting with ST-segment-elevation myocardial infarction and non-ST-segment-elevation myocardial infarction. Approach and Results: We identified independent predictors of platelet activation through certain receptors and a murine MI model further explored these findings. Platelets from healthy women and female mice are more reactive through PARs (protease-activated receptors) compared with platelets from men and male mice. Multivariate regression analyses revealed male sex and non-ST-segment-elevation myocardial infarction as independent predictors of enhanced PAR1 activation in human platelets. Platelet PAR1 signaling decreased in women and increased in men during MI which was the opposite of what was observed during healthy conditions. Similarly, in mice, thrombin-mediated platelet activation was greater in healthy females compared with males, and lesser in females compared with males at the time of MI. CONCLUSIONS: Sex-specific signaling in platelets seems to be a cross-species phenomenon. The divergent platelet phenotype in males and females at the time of MI suggests a sex-specific antiplatelet drug regimen should be prospectively evaluated.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Region-specific proteolysis differentially modulates type 2 and type 3 inositol 1,4,5-trisphosphate receptor activity in models of acute pancreatitis.

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IP3R) by a diverse array of regulatory inputs results in intracellular Ca2+ signals with distinct characteristics. These events allow the activation of specific downstream effectors. We reported previously that region-specific proteolysis represents a novel regulatory event for type 1 IP3R (R1). Specifically, caspase-fragmented R1 display a marked increase in single-channel open probability. More importantly, the distinct characteristics of the Ca2+ signals elicited via fragmented R1 can activate alternate downstream effectors. In this report, we expand these studies to investigate whether all IP3R subtypes are regulated by proteolysis. We now show that type 2 and type 3 IP3R (R2 and R3, respectively) are proteolytically cleaved in rodent models of acute pancreatitis. Surprisingly, fragmented IP3R retained tetrameric architecture, remained embedded in endoplasmic reticulum membranes and were not functionally disabled. Proteolysis was associated with a marked attenuation of the frequency of Ca2+ signals in pancreatic lobules. Consistent with these data, expression of DNAs encoding complementary R2 and R3 peptides mimicking fragmented receptors at particular sites, resulted in a significant decrease in the frequency of agonist-stimulated Ca2+ oscillations. Further, proteolysis of R2 resulted in a marked decrease in single-channel open probability. Taken together, proteolytic fragmentation modulates R2 and R3 activity in a region-specific manner, and this event may contribute to the altered Ca2+ signals in pancreatic acinar cells during acute pancreatitis.

Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

The inositol 1,4,5 trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP3R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP3R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca2+ release profile and protein kinase A modulation of Ca2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP3R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP3R1 may represent a novel form of modulation of IP3R1 channel function and increases the repertoire of Ca2+ signals achievable through this channel.

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs.

The ability of inositol 1,4,5-trisphosphate receptors (IP3R) to precisely initiate and generate a diverse variety of intracellular Ca(2+) signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP3, Ca(2+), and ATP). However, the contribution of IP3R heterotetramerization to Ca(2+) signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca(2+) signals. Under conditions where key regulators of IP3R function are optimal for Ca(2+) release, we demonstrate that individual monomers within heteromeric IP3Rs contributed equally toward generating a distinct 'blended' sensitivity to IP3 that is likely dictated by the unique IP3 binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP3R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP3R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP3-induced Ca(2+) signaling in cells expressing multiple IP3R isoforms.

Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second high-affinity open state.

Local anesthetics (LAs) block resting, open, and inactivated states of voltage-gated Na(+) channels where inactivated states are thought to bind with highest affinity. However, reports of fast-onset block occurring over milliseconds hint at high-affinity block of open channels. Movement of voltage-sensor domain IV-segment 4 (DIVS4) has been associated with high affinity LA block termed voltage-sensor block (VSB) that also leads to a second open state. These observations point to a second high-affinity open state that may underlie fast-onset block. To test for this state, we analyzed the modulation of Na(+) currents by lidocaine and its quaternary derivative (QX222) from heterologously expressed (Xenopus laevis oocytes) rat skeletal muscle µ1 NaV1.4 (rSkM1) with ß1 (WT-ß1), and a mutant form (IFM-QQQ mutation in the III-IV interdomain, QQQ) lacking fast inactivation, in combination with Markov kinetic gating models. 100 µM lidocaine induced fast-onset (τonset≈2 ms), long-lived (τrecovery≈120 ms) block of WT-ß1 macroscopic currents. Lidocaine blocked single-channel and macroscopic QQQ currents in agreement with our previously described mechanism of dual, open-channel block (DOB mechanism). A DOB kinetic model reproduced lidocaine effects on QQQ currents. The DOB model was extended to include trapping fast-inactivation and activation gates, and a second open state (OS2); the latter arising from DIVS4 translocation that precedes inactivation and exhibits high-affinity, lidocaine binding (apparent Kd=25 µM) that accords with VSB (DOB-S2VSB mechanism). The DOB-S2VSB kinetic model predicted fast-onset block of WT-ß1. The findings support the involvement of a second, high-affinity, open state in lidocaine modulation of Na(+) channels.

The BRCA1 tumor suppressor binds to inositol 1,4,5-trisphosphate receptors to stimulate apoptotic calcium release.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Tetrameric, active PKM2 inhibits IP3 receptors, potentially requiring GRP75 as an additional interaction partner.

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP3R). This interaction suppresses IP3R-mediated cytosolic [Ca2+] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IP3Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP3R-mediated Ca2+ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2C424L, a tetrameric, highly active PKM2 mutant, but not inactive PKM2K270M or the less active PKM2K305Q, suppressed IP3R-mediated Ca2+ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP3R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP3R1), the presumed interaction sites of PKM2 on the IP3R. Moreover, on-nucleus patch clamp of heterologously expressed IP3R1 in DT40 cells devoid of endogenous IP3Rs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IP3R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP3R1, IP3R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP3R, but also PKM2:GRP75 and GRP75:IP3R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca2+ signaling via the IP3R through a multiprotein complex involving GRP75.

Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity.

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) activity resulting in modulation of intracellular Ca 2+ release. This regulation is thought to define the spatio-temporal patterns of Ca 2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP 3 and Ca 2+ are obligatory for IP 3 R channel opening, however, Ca 2+ regulates IP 3 R activity in a biphasic manner. Mutational studies have revealed that Ca 2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP 3 R channel opening without altering the Ca 2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca 2+ binding site is responsible for the reduction in IP 3 R activity at higher [Ca 2+ ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca 2+ in cryo-EM structures of the IP 3 R type 3. This site is termed the "CD Ca 2+ binding site" and is well-conserved in all IP 3 R sub-types. We show that the CD site Ca 2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP 3 -generating agonists. Ca 2+ binding mutants displayed spontaneous elemental Ca 2+ events (Ca 2+ puffs) and the number of IP 3 -induced Ca 2+ puffs was significantly augmented in cells stably expressing Ca 2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca 2+ ] on single channel-open probability (P o ) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca 2+ binding to the putative CD Ca 2+ inhibitory site facilitates the reduction in IP 3 R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca 2+ binding motifs may contribute to the biphasic regulation of IP 3 -induced Ca 2+ release.

Dysregulated Ca2+ signaling, fluid secretion, and mitochondrial function in a mouse model of early Sjögren's syndrome.

Saliva is essential for oral health. The molecular mechanisms leading to physiological fluid secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of inflammatory immune cell infiltration within the salivary glands and glandular hypofunction. In this study, we investigated in a mouse model system, mechanisms of glandular hypofunction caused by the activation of the stimulator of interferon genes (STING) pathway. Glandular hypofunction and SS-like disease were induced by treatment with 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), a small molecule agonist of murine STING. Contrary to our expectations, despite a significant reduction in fluid secretion in DMXAA-treated mice, in vivo imaging demonstrated that neural stimulation resulted in greatly enhanced spatially averaged cytosolic Ca2+ levels. Notably, however, the spatiotemporal characteristics of the Ca2+ signals were altered to signals that propagated throughout the entire cytoplasm as opposed to largely apically confined Ca2+ rises observed without treatment. Despite the augmented Ca2+ signals, muscarinic stimulation resulted in reduced activation of TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. However, super-resolution microscopy revealed a disruption in the intimate colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels in relation to TMEM16a. TMEM16a channel activation was also reduced when intracellular Ca2+ buffering was increased. These data are consistent with altered local coupling between the channels contributing to the reduced activation of TMEM16a. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics and secretion is an energetically expensive process. Disrupted mitochondrial morphology, a depolarized mitochondrial membrane potential, and reduced oxygen consumption rate were observed in DMXAA-treated animals compared to control animals. We report that early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction and likely the progression of SS disease.

Loss of STIM1 and STIM2 in salivary glands disrupts ANO1 function but does not induce Sjogren's disease.

Sjogren's disease (SjD) is an autoimmune disease characterized by xerostomia (dry mouth), lymphocytic infiltration into salivary glands and the presence of SSA and SSB autoantibodies. Xerostomia is caused by hypofunction of the salivary glands and has been involved in the development of SjD. Saliva production is regulated by parasympathetic input into the glands initiating intracellular Ca 2+ signals that activate the store operated Ca 2+ entry (SOCE) pathway eliciting sustained Ca 2+ influx. SOCE is mediated by the STIM1 and STIM2 proteins and the ORAI1 Ca 2+ channel. However, there are no studies on the effects of lack of STIM1/2 function in salivary acini in animal models and its impact on SjD. Here we report that male and female mice lacking Stim1 and Stim2 ( Stim1/2 K14Cre ) in salivary glands showed reduced intracellular Ca 2+ levels via SOCE in parotid acini and hyposalivate upon pilocarpine stimulation. Bulk RNASeq of the parotid glands of Stim1/2 K14Cre mice showed a decrease in the expression of Stim1/2 but no other Ca 2+ associated genes mediating saliva fluid secretion. SOCE was however functionally required for the activation of the Ca 2+ activated chloride channel ANO1. Despite hyposalivation, ageing Stim1/2 K14Cre mice showed no evidence of lymphocytic infiltration in the glands or elevated levels of SSA or SSB autoantibodies in the serum, which may be linked to the downregulation of the toll-like receptor 8 ( Tlr8 ). By contrast, salivary gland biopsies of SjD patients showed increased STIM1 and TLR8 expression, and induction of SOCE in a salivary gland cell line increased the expression of TLR8 . Our data demonstrate that SOCE is an important activator of ANO1 function and saliva fluid secretion in salivary glands. They also provide a novel link between SOCE and TLR8 signaling which may explain why loss of SOCE does not result in SjD.

A kinetic model for type I and II IP3R accounting for mode changes.

Based upon an extensive single-channel data set, a Markov model for types I and II inositol trisphosphate receptors (IP(3)R) is developed. The model aims to represent accurately the kinetics of both receptor types of IP(3)R depending on the concentrations of inositol trisphosphate (IP(3)), adenosine trisphosphate (ATP), and intracellular calcium (Ca(2+)). In particular, the model takes into account that for some combinations of ligands the IP(3)R switches between extended periods of inactivity alternating with intervals of bursting activity (mode changes). In a first step, the inactive and active modes are modeled separately. It is found that, within modes, both receptor types are ligand-independent. In a second step, the submodels are connected by transition rates. Ligand-dependent regulation of the channel activity is achieved by modulating these transitions between active and inactive modes. As a result, a compact representation of the IP(3)R is obtained that accurately captures stochastic single-channel dynamics including mode changes in a model with six states and 10 rate constants, only two of which are ligand-dependent.

Differential regulation of the InsP3 receptor type-1 and -2 single channel properties by InsP3, Ca²âº and ATP.

An elevation of intracellular Ca2+ levels as a result of InsP3 receptor (InsP3R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP3R activity is tightly controlled by the levels of the primary ligands, InsP3, Ca2+ and ATP. Importantly, InsP3Rs are regulated by Ca2+ i in a biphasic manner. Ca2+ release through all InsP3R family members is also modulated dramatically by ATP, albeit with sub-type-specific properties. To ascertain if a common mechanism can account for ATP and Ca2+ regulation of these InsP3R family members, we examined the effects of [ATP] on the Ca2+ dependency of rat InsP3R-1 (rInsP3R-1) and mouse InsP3R-2 (mInsP3R-2) activity expressed in DT40-3KO cells. We used the on-nucleus patch clamp recording technique with various [ATP], [InsP3] and [Ca2+] in the patch pipette and measured single InsP3R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP3] and [ATP], the activity of rInsP3R-1 and mInsP3R-2 was essentially identical in terms of single channel conductance, maximal achievable open probability (Po) and the [Ca2+] required for activation and inhibition of activity. However, in contrast to rInsP3R-1 at saturating [InsP3], the activity of mInsP3R-2 was unaffected by [ATP]. At lower [InsP3], ATP had dramatic effects on mInsP3R-2 Po, but unlike the rInsP3R-1, this did not occur by altering the relative Ca2+ dependency, but by simply increasing the maximally achievable Po at a particular [InsP3] and [Ca2+]. [InsP3] did not alter the biphasic regulation of activity by Ca2+ in either rInsP3R-1 or mInsP3R-2. Analysis of the single channel kinetics indicated that Ca2+ and ATP modulate the Po predominately by facilitating extended bursting activity of the channel but the underlying biophysical mechanism appears to be distinct for each receptor. Subtype-specific regulation of InsP3R channel activity probably contributes to the fidelity of Ca2+ signalling in cells expressing these receptor subtypes.

Segregated cation flux by TPC2 biases Ca2+ signaling through lysosomes.

Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P2. Whereas NAADP rendered the channel Ca2+-permeable and PI(3,5)P2 rendered the channel Na+-selective, a combination of the two increased Ca2+ but not Na+ flux. Mechanistically, this was due to an increase in Ca2+ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P2 mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca2+ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca2+ signaling.

Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL's inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL's anti-apoptotic function.

Platelet olfactory receptor activation limits platelet reactivity and growth of aortic aneurysms.

As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. Although it has been demonstrated that antiplatelet drugs suppress the growth of abdominal aortic aneurysms (AAA) in patients, we found that a certain degree of platelet reactivity persisted in spite of aspirin therapy, urging us to consider additional antiplatelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors, and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13, which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies revealed that olfactory receptors regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.