Depletion Of Calcium Stores Research Articles

Peroxisomes contribute to intracellular calcium dynamics in cardiomyocytes and non-excitable cells.

Peroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Furthermore, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes can take up calcium. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected both in HeLa cells and in cardiomyocytes. Cardiac peroxisomes take up calcium on beat-to-beat basis. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics of cardiomyocytes.

Role of STIM2 and Orai proteins in regulating TRPC1 channel activity upon calcium store depletion

Store-operated calcium channels are the major player in calcium signaling in non-excitable cells. Store-operated calcium entry is associated with the Orai, stromal interaction molecule (STIM), and transient receptor potential canonical (TRPC) protein families. Researchers have provided conflicting data about TRPC1 channel regulation by Orai and STIM. To determine how Orai and STIM influence endogenous TRPC1 pore properties and regulation, we used single channel patch-clamp recordings. Here we showed that knockout or knockdown of Orai1 or Orai3 or overexpression of the dominant-negative mutant Orai1 E106Q did not change the conductance or selectivity of single TRPC1 channels. In addition, these TRPC1 channel properties did not depend on the amount of STIM1 and STIM2 proteins. To study STIM2-mediated regulation of TRPC1 channels, we utilized partial calcium store depletion induced by application of 10 nM thapsigargin (Tg). TRPC1 activation by endogenous STIM2 was greatly decreased in acute extracellular calcium-free experiments. STIM2 overexpression increased both the basal activity and number of silent TRPC1 channels in the plasma membrane. After calcium store depletion, overexpressed STIM2 directly activated TRPC1 in the plasma membrane even without calcium entry in acute experiments. However, this effect was abrogated by co-expression with the non-permeable Orai1 E106Q mutant protein. Taken together, our single-channel patch clamp experiments clearly demonstrated that endogenous TRPC1 forms a channel pore without involving Orai proteins. Calcium entry through Orai triggered TRPC1 channel activation in the plasma membrane, while subsequent STIM2-mediated TRPC1 activity regulation was not dependent on calcium entry.

Antidiuretic hormone inhibits osteogenic differentiation of dental follicle stem cells via V1a receptors and the PLC-IP3 pathway

ObjectiveThe aim of this study was to elucidate the molecular mechanism by which antidiuretic hormone (ADH) inhibited osteogenesis in dental follicle stem cells. DesignRat dental follicle stem cells were cultured in osteogenic differentiation medium supplemented with ADH. Alkaline phosphatase enzyme activity, Alizarin Red S staining, MTT assay and RT-qPCR was used to examine ADH’s impact on cell mineralization, viability, and osteogenic gene expression. Real-time calcium imaging analysis was performed to identify the ADH receptor and its mechanism of action. ResultsADH supplementation to the osteogenic differentiation medium inhibited cell mineralization without compromising cell viability and downregulated the expression of key osteogenic genes: DCN (Decorin), RUNX2 (Runt-related transcription factor 2) and BSP (Bone sialoprotein). Real-time calcium imaging analysis revealed that ADH (1−1000 nM) increased intracellular calcium in a concentration-dependent manner. Pretreatment of cells with V2255, a V1a receptor blocker, inhibited the calcium signals, but not with the V1b (Nelivaptan) or V2 (Tolvaptan). V2255 also reversed the inhibitory effect of ADH on osteogenesis. Furthermore, U73122, a Phospholipase C (PLC) inhibitor, 2-APB, an Inositol Triphosphate (IP3) receptor blocker, and depletion of endoplasmic reticulum calcium stores abolished the calcium signals by ADH. ConclusionsOur results demonstrated that ADH activates V1a receptors and the PLC-IP3 pathway to stimulate intracellular calcium signals, which inhibits cell mineralization and osteogenic gene expression. These findings uncovered a novel function for ADH as a negative regulator of osteogenesis in dental follicle stem cells. The role of ADH in the pathogenesis of bone diseases remains to be determined.

Role of Internal Ca2+ Stores in KCa Channel Feedback Control of Human Uterine Contractions

Background Minute-to-minute regulation of intracellular calcium concentrations is essential to allow a laboring uterus to phasically contract and relax in a manner required for delivery. Cytosolic calcium levels increase through influx of calcium through L-type calcium channels and are amplified by SR calcium release through inositol 1, 4, 5-triphosphate receptor (IP3R) channels and calcium induced calcium release. A specific class of small conductance calcium-activated potassium (KCa2.3) channels has been strongly implicated in the negative feedback control of membrane potential and intracellular calcium levels. Uterine contractions can be suppressed by amplifying this calcium-dependent feedback system. CyPPA, a positive modulator of KCa2.3, reduces contraction amplitudes in human and rodent tissue. Previous studies of endothelial and neuronal cells suggest focused Ca2+ release from internal stores in close proximity to the plasma membrane, may play an important role in KCa channel control. Indeed, close proximity and interaction of endoplasmic reticulum Ca2+ release channels (e.g. IP3Rs) and KCa channels may regulate intracellular calcium in a variety of tissues. This study aims to assess whether internal stores contribute to KCa2.3 dependent feedback regulation of contraction in the human myometrium. Methods Myograph studies were performed on human uterine tissues collected from non-pregnant patients undergoing hysterectomy. Uterine strips were sutured end-to-end to form rings, mounted onto myograph pins, and allowed to develop spontaneous phasic contractions. Concentration-dependent suppression of contractions with the positive KCa2.3 modulator CyPPA (3 μM, 10 μM, 30 μM) was assessed following depletion of internal calcium stores (using the SERCA inhibitor cyclopiazonic acid, CPA) or following treatment with the vehicle DMSO (controls). Results Utilizing myographical recordings and LabChart software, contraction area under curve (AUC) was calculated and normalized to time. Addition of 3 μM CyPPA resulted in a control group mean AUC of 1.287 mN/min (± 0.417) and 2.261 mN/min (± 1.338) for the treatment group [n=5]. The mean AUC with 10 μM CyPPA was 1.129 mN/min (± 0.571) for the control group and 2.164 mN/min (± 1.387) for the treatment group. After 30 μM CyPPA the mean AUC for the control group was 0.734 mN/min (± 0.471) and 1.863 mN/min (± 1.364) for the treatment group. Our group found that CPA pretreated uterine tissue had a significantly higher AUC than the control group (p<0.5), after addition of 30 μM CyPPA. Further analysis demonstrated no significant difference with CyPPA concentrations of 3 and 10 μM, but did reveal a trend of reduced AUC in the control group compared to the treatment group. These findings suggest blocking SERCA and inhibiting SR reuptake of calcium may decrease CyPPAs ability to diminish contractions, especially at higher concentrations. Conclusions Phasic contractions of isolated human uterine strips vary considerably with respect to amplitude and frequency. Overall, the findings suggest internal calcium store release may play an important role in moderating uterine contractions and augmenting KCa2.3 channel suppression of uterine contractility. Extended studies will assess whether this mechanism can be exploited as an effective tocolytic strategy.

Abstract 14219: Oxidation of PKA-RIα Protects Against Ischemia-reperfusion Injury by Inhibiting Lysosomal-triggered Calcium Release

Introduction: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, the regulatory Iα subunit of Protein Kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulfide bonds within the holoenzyme complex. However, the impact of disulfide formation on kinase function, and its influence on PKA signaling in the context of heart disease remains unknown. Methods &amp; Results: Myocardial ischemia-reperfusion (I/R) was found to be a potent inducer of PKARIα disulfide formation in vivo , both in mice and in humans. Using imaging modalities with high spatial and temporal resolution, we found that this conformation did not increase intrinsic PKA catalytic activity, but rather facilitated enhanced AKAP-dependent compartmentation of PKARIα in the adult mouse left ventricular (LV) myocyte, with preferential localization to the lysosome under oxidized conditions (n=38-41 myocytes, N=3 animals, p&lt;0.01). Investigations in isolated LV myocytes revealed disulfide-modified PKARIα to be a significant regulator of lysosomal two pore channel (TPC)-dependent calcium-induced calcium release, with myocytes from ‘redox dead’ PKARIα mice (Cys17Ser) displaying spontaneous sarcoplasmic reticulum calcium release events and pronounced intracellular calcium oscillations. These events were prevented by ryanodine receptor blockade (1 mM tetracaine; n=14, p&lt;0.01), acute depletion of lysosomal calcium stores (100 nM bafilomycin; n=7; p&lt;0.01), or TPC inhibition (5 μM Ned-19; n=9; p&lt;0.05). Absence of I/R-induced disulfide formation in “redox dead” PKARIα mouse hearts resulted in larger infarcts (2-fold increase, p&lt;0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p&lt;0.001), which could be fully prevented by administering the TPC inhibitor, Ned-19, at the time of reperfusion. Conclusions: Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKA oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics.

Orai-1 and Orai-2 regulate oral cancer cell migration and colonisation by suppressing Akt/mTOR/NF-κB signalling

Despite remarkable progress in understanding and treating oral cancer (OC), it still remains one of the life-threatening diseases and predominant cancers in the world. Therefore, deciphering the molecular mechanisms of this disease would help us to develop highly efficacious therapies. Multiple lines of evidence suggest that calcium and its dysregulation play significant role in the development of various cancers. As an adaptation of survival mechanism, upon depletion of ER calcium stores, store-operated calcium entry (SOCE) has been induced via SOCE channels (SOCC) in various mammalian cells. SOCC are regulated by Orai-1, Orai-2 and Orai-3 located on plasma membrane and two calcium-sensing ER membrane proteins known as stromal interaction molecules (STIM-1 and STIM-2). Hence, the present study was aimed at analysing the role of Orai-1 and Orai-2 in oral cancer and the underlying mechanism. Our results suggest that both Orai-1 and Orai-2 proteins were overexpressed in oral cancer tissues and cell lines (SAS) compared to normal epithelial tissues and cell lines respectively. In addition, silencing of Orai-1 and Orai-2 via chemical SOCE inhibitors and siRNAs inhibited calcium uptake and suppressed oral cancer cell proliferation, colony formation and migration. Furthermore, silencing of Orai-1 and Orai-2 inhibited Akt/mTOR/NF-κB pathway in oral cancer cells. Interestingly, tobacco carcinogen NNN and synthetic carcinogen 4-NQO, enhanced the expression of Orai-1 and Orai-2 in SAS cells. Therefore, we conclude that Orai-1 and Orai-2 have significant role in oral cancer and can be further explored to develop novel therapies for the treatment of this disease.

Selenoprotein T: An Essential Oxidoreductase Serving as a Guardian of Endoplasmic Reticulum Homeostasis.

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N-glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases.

Distribution of Acid Sensing Ion Channels in Axonal Growth Cones and Presynaptic Membrane of Cultured Hippocampal Neurons.

Although acid-sensing ion channels (ASICs) are widely expressed in the central nervous system, their distribution and roles in axonal growth cones remain unclear. In this study, we examined ASIC localization and function in the axonal growth cones of cultured immature hippocampal neurons. Our immunocytochemical data showed that native and overexpressed ASIC1a and ASIC2a are both localized in growth cones of cultured young hippocampal neurons. Calcium imaging and electrophysiological assay results were utilized to validate their function. The calcium imaging test results indicated that the ASICs (primarily ASIC1a) present in growth cones mediate calcium influx despite the addition of voltage-gated Ca2+ channels antagonists and the depletion of intracellular calcium stores. The electrophysiological tests results suggested that a rapid decrease in extracellular pH at the growth cones of voltage-clamped neurons elicits inward currents that were blocked by bath application of the ASIC antagonist amiloride, showing that the ASICs expressed at growth cones are functional. The subsequent immuno-colocalization test results demonstrated that ASIC1a and ASIC2a are both colocalized with Neurofilament-H and Bassoon in mature hippocampal neurons. This finding demonstrated that after reaching maturity, ASIC1a and ASIC2a are both distributed in axons and the presynaptic membrane. Our data reveal the distribution of functional ASICs in growth cones of immature hippocampal neurons and the presence of ASICs in the axons and presynaptic membrane of mature hippocampal neurons, indicating a possible role for ASICs in axonal guidance, synapse formation and neurotransmitter release.

The role of sphingolipids in endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.

MO059CONTRIBUTION OF OPIOID RECEPTOR SIGNALING IN PODOCYTES TOWARDS THE DEVELOPMENT OF SALT-SENSITIVE HYPERTENSION AND KIDNEY INJURY

Abstract Background and Aims The global increase in opioid use requires practitioners to be aware of the side effects and contraindications of these medications in order to avoid complications, especially in patients with organ dysfunction such as cardiovascular disease and chronic kidney disease. It has been reported that opioid use is strongly associated with predictors of poor cardiovascular outcomes such as increased albuminuria and reduced GFR. Opioids work by binding to kappa-, mu- and delta-opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. Method To test this hypothesis, we applied specific OR agonists to: 1) in vitro - immortalized cultured human podocytes (hPod), 2) ex vivo - freshly isolated human and rat glomeruli; and 3) in vivo - Dahl salt-sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using calcium fluorescent dyes. For chronic studies Dahl SS rats were fed a high salt (HS; 8%) NaCl diet for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Results The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 µM) in a 2 mM calcium solution. A pre-application of kappa-OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the calcium-free solution or a pre-application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa-OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre-application of SAR7334. Effect of kappa-OR agonist was specific since application of Damgo (mu-OR agonist) or DPDPE (delta-OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL-treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Alb/Cre at day 7 of HS), higher mean arterial pressures (179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Cre) compared to control rats. Glomeruli isolated from the hypertensive opioid treated rats had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) in podocytes compared to non-treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Conclusion Our data establish that stimulation of kappa-ORs are specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which causes calcium overload and cellular damage. Observed increase in the calcium flux in podocytes most likely further contribute to the kidney injury and progression of salt-induced hypertension.

The Role of Opioid Receptors in Podocyte Injury and Kidney Damage During the Development of Salt‐Induced Hypertension

The global increase in opioid use requires to better understand the side effects of opioids in order to avoid additional complications. Opioids are common analgesics for patients with hypertension and CKD despite its association with increased albuminuria and reduced glomerular filtration rate. Opioids work by binding to kappa‐, mu‐ and delta‐opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. To test this hypothesis, we applied specific OR agonists to: 1) in vitro ‐ immortalized cultured human podocytes (hPod), 2) ex vivo ‐ freshly isolated human and rat glomeruli; and 3) in vivo ‐ Dahl salt‐sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using Fluo4/FuraRed and Fluo8 fluorescent dyes. For chronic studies Dahl SS rats were on a 0.4% (LS) or 8% (HS) NaCl diets for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa‐OR agonist. The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 μM) in a 2 mM calcium solution. A pre‐application of kappa‐OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the 0 mM calcium solution or a pre‐application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa‐OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre‐application of SAR7334. Effect of kappa‐OR agonist was specific since application of Damgo (mu‐OR agonist) or DPDPE (delta‐OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL‐treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Albumin/Creatinine at day 7 of HS), higher mean arterial pressures (MAP, 179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Creatinine) compared to control rats. Podocytes isolated from the glomeruli from the hypertensive opioid treated animals had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) compared to non‐treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Our data established that stimulation of kappa‐ORs were specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which caused calcium overload and cellular damage. The opioid‐induced increase in the calcium flux in podocytes is expected to contribute to podocytopathy, proteinuria, kidney injury and progression of salt‐induced hypertension.

TRPC5 regulates axonal outgrowth in developing retinal ganglion cells

The TRPC5 ion channel is activated upon depletion of intracellular calcium stores, as well as by various stimuli such as nitric oxide (NO), membrane stretch, and cold temperatures. TRPC5 is abundantly expressed in the central nervous system where it has important neuronal functions. In the chick retina, TRPC5 expression was shown to be restricted to amacrine cells (ACs) and Müller glial cells, although its expression was also observed in the ganglion cell layer (GCL) in displaced ACs, as determined by their characteristic cell morphology. However, it is possible that this expression analysis alone might be insufficient to fully understand the expression of TRPC5 in retinal ganglion cells (RGCs). Hence, we analyzed TRPC5 expression by in situ hybridization and immunostaining in the developing mouse retina, and for the first time identified that developing and mature RGCs strongly express TRPC5. The expression begins at E14.5, and is restricted to ACs and RGCs. It was reported that TRPC5 negatively regulates axonal outgrowth in hippocampal neurons. We thus hypothesized that TRPC5 might have similar functions in RGCs since they extend very long axons toward the brain, and this characteristic significantly differs from other retinal cell types. To elucidate its possible involvement in axonal outgrowth, we inhibited TRPC5 activity in developing RGCs which significantly increased RGC axon length. In contrast, overexpression of TRPC5 inhibited axonal outgrowth in developing RGCs. These results indicate that TRPC5 is an important negative regulator of RGC axonal outgrowth. Since TRPC5 is a mechanosensor, it might function to sense abnormal intraocular pressure changes, and could contribute to the death of RGCs in diseases such as glaucoma. In this case, excessive Ca2+ entry through TRPC5 might induce dendritic and axonal remodeling, which could lead to cell death, as our findings clearly indicate that TRPC5 is an important regulator of neurite remodeling.

Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons.

The striatum plays an important role in linking cortical activity to basal ganglia output. Striatal neurons exhibit spontaneous slow Ca2+ oscillations that result from Ca2+ release from the endoplasmic reticulum (ER) induced by the mGluR5-IP3R signaling cascade. The maximum duration of a single oscillatory event is about 300 s. A major question arises as to how such a long-duration Ca2+ elevation is maintained. Store-operated calcium channels (SOCCs) are one of the calcium (Ca2+)-permeable ion channels. SOCCs are opened by activating the metabotropic glutamate receptor type 5 and inositol 1,4,5-trisphosphate receptor (mGluR5-IP3R) signal transduction cascade and are related to the pathophysiology of several neurological disorders. However, the functions of SOCCs in striatal neurons remain unclear. Here, we show that SOCCs exert a functional role in striatal GABAergic neurons. Depletion of calcium stores from the ER induced large, sustained calcium entry that was blocked by SKF96365, an inhibitor of SOCCs. Moreover, the application of SKF96365 greatly reduced the frequency of slow Ca2+ oscillations. The present results indicate that SOCCs contribute to Ca2+ signaling in striatal GABAergic neurons, including medium spiny projection neurons (MSNs) and GABAergic interneurons, through elevated Ca2+ due to spontaneous slow Ca2+ oscillations.

Inhibiting store-operated calcium entry attenuates white matter secondary degeneration following SCI

Axonal degeneration plays a key role in the pathogenesis of numerous neurological disorders including spinal cord injury. After the irreversible destruction of the white matter elements during the primary (mechanical) injury, spared axons and their supporting glial cells begin to breakdown causing an expansion of the lesion site. Here we mechanistically link external sources of calcium entry through axoplasmic reticulum calcium store depletion that contributes to secondary axonal degeneration through a process called store-operated calcium entry. There is increasing evidence suggesting that store-operated calcium entry impairment is responsible for numerous disorders. Nevertheless, its role following spinal cord injury remains poorly understood. We hypothesize that store-operated calcium entry mediates secondary white matter degeneration after spinal cord injury. We used our previously published model of laser-induced spinal cord injury to focally transect mid cervical dorsal column axons from live 6–8-week-old heterozygous CNPaseGFP/+: Thy1YFP+ double transgenic murine spinal cord preparations (five treated, eight controls) and documented the dynamic changes in axons over time using two-photon excitation microscopy. We report that 1 hour delayed treatment with YM-58483, a potent inhibitor of store-operated calcium entry, significantly decreased intra-axonal calcium accumulation, axonal dieback both proximal and distal to the lesion site, reduced secondary axonal “bystander” damage acutely after injury, and promoted greater oligodendrocyte survival compared to controls. We also targeted store-operated calcium entry following a clinically relevant contusion spinal cord injury model in vivo. Adult, 6–8-week-old Advillin-Cre: Ai9 mice were subjected to a mild 30 kdyn contusion and imaged to observe secondary axonal degeneration in live animals. We found that delayed treatment with YM-58483 increased axonal survival and reduced axonal spheroid formation compared to controls (n = 5 mice per group). These findings suggest that blocking store-operated calcium entry acutely is neuroprotective and introduces a novel target to prevent pathological calcium entry following spinal cord injury using a clinically relevant model.

Subtype specific targeting of calcium signaling in breast cancer

An important component of breast milk, calcium also appears as radiographically prominent microcalcifications in breast tissue that are often the earliest sign of malignancy. Ionic Ca2+ is a universal second messenger that controls a wide swathe of effector pathways integral to gene transcription, cell cycle control, differentiation, proliferation, cell migration, and apoptosis. Whereas prolonged elevation in resting Ca2+ levels drives proliferation to initiate and sustain tumor growth, depletion of calcium stores and attenuation of calcium influx pathways underlies tumor chemoresistance and evasion of apoptosis. This paradox of Ca2+ homeostasis highlights the challenge of targeting Ca2+ signaling pathways for breast cancer therapy. Furthermore, breast cancer is a heterogeneous disease classified into distinct subtypes based on tumor origin, stage of invasiveness and hormone receptor status. Classification is important for tailoring treatment, and in predicting clinical outcome or response to chemotherapy. There have been numerous reports of dysregulated expression, localization or activity of Ca2+ channels, regulators and pumps in breast cancer. An important aspect of these alterations is that they are specific to breast cancer subtype, as exemplified by a reciprocal switch in secretory pathway Ca2+-ATPase isoforms SPCA1 and SPCA2 depending on receptor status. In this review, we discuss the current knowledge of subtype specific changes in calcium channels and pumps, with a focus on functional insights that may inform new opportunities for breast cancer therapy.

A Statistical View on Calcium Oscillations.

Transient rises and falls of the intracellular calcium concentration have been observed in numerous cell types and under a plethora of conditions. There is now a growing body of evidence that these whole-cell calcium oscillations are stochastic, which poses a significant challenge for modelling. In this review, we take a closer look at recently developed statistical approaches to calcium oscillations. These models describe the timing of whole-cell calcium spikes, yet their parametrisations reflect subcellular processes. We show how non-stationary calcium spike sequences, which e.g. occur during slow depletion of intracellular calcium stores or in the presence of time-dependent stimulation, can be analysed with the help of so-called intensity functions. By utilising Bayesian concepts, we demonstrate how values of key parameters of the statistical model can be inferred from single cell calcium spike sequences and illustrate what information whole-cell statistical models can provide about the subcellular mechanistic processes that drive calcium oscillations. In particular, we find that the interspike interval distribution of HEK293 cells under constant stimulation is captured by a Gamma distribution.

2161Redox-mediated PKA-RIalpha localisation to the lysosome inhibits myocardial calcium release and robustly reduces myocardial injury

Abstract Background Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulphide bonds within the holoenzyme complex. However, the effect of PKARIα oxidation on downstream signalling in the heart, particularly under states of oxidative stress, remains unexplored. Purpose To determine the direct functional consequences of PKARIα oxidation in the heart and investigate their impact on ischaemia/reperfusion (I/R) injury. Methods and results Experiments using the AKAR3ev FRET biosensor in murine left ventricular (LV) myocytes and Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged wild-type (WT) and mutant RIα proteins expressed in RIα-null fibroblasts showed that PKARIα oxidation does not increase the kinases' catalytic activity, but enhances its binding to A-kinase anchoring proteins (AKAP; n=30–39/N=3, p&lt;0.01). Super-resolution microscopy revealed localisation of oxidised PKARIα to lysosomes in WT myocytes, which was completely absent in “redox dead” Cys17Ser PKARIα knock-in mice (KI; panel A; n=38–41/N=3, p&lt;0.01) and reduced when AKAP binding was prevented using the RIAD disruptor peptide (30.6±5.1% reduction; n=35–37/N=3, p&lt;0.01). Displacement of PKARIα from lysosomes resulted in spontaneous sarcoplasmic reticulum calcium release and dramatic calcium oscillations in KI LV myocytes (panel B), which were preventable by ryanodine receptor blockade (1 mM tetracaine; n=14, p&lt;0.01), acute depletion of endolysosomal calcium stores (100 nM bafilomycin; n=7; p&lt;0.01), or lysosomal two-pore channel inhibition (5 μM Ned-19; n=9; p&lt;0.05). I/R (secondary to cardiopulmonary bypass) was found to induce PKARIα oxidation in the myocardium of patients undergoing cardiac surgery (panel C; n=18, p&lt;0.05). Absence of this response in KI mouse hearts resulted in 2-fold larger infarcts (p&lt;0.01) and a concomitant reduction in LV contractile recovery (final LVDP of 55.9±8.6 vs 82.5±7.1 mmHg in WT; n=7–8, p&lt;0.05), both which were prevented by addition of Ned-19 at the time of reperfusion (panel D; n=4, p&lt;0.01). Conclusions Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKARIα oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics. Acknowledgement/Funding British Heart Foundation CH/12/3/29609, RG/16/12/32451; Garfield-Weston Foundation MPS/IVIMS-11/12-4032; Wellcome Trust Fellowship 0998981Z/12/Z

Loss of Orai2-Mediated Capacitative Ca2+ Entry Is Neuroprotective in Acute Ischemic Stroke.

Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca2+ entry, which is triggered by the depletion of intracellular calcium stores, contributes to ischemia-induced calcium influx in neurons, but the responsible Ca2+ channel is not known. Methods- Here, we have generated mice lacking the calcium channel subunit Orai2 and analyzed them in experimental stroke. Results- Orai2-deficient mice were protected from ischemic neuronal death both during acute ischemia under vessel occlusion and during ischemia/reperfusion upon successful recanalization. Calcium signals induced by calcium store depletion or oxygen/glucose deprivation were significantly diminished in Orai2-deficient neurons demonstrating that Orai2 is a central mediator of neuronal capacitative Ca2+ entry and is involved in calcium overload during ischemia. Conclusions- Our experimental data identify Orai2 as an attractive target for pharmaceutical intervention in acute stroke.

Transient Receptor Potential channels (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Transient Receptor Potential channels (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Probing the Mechanisms by which Septins Regulate ORAI1 Function

The STIM-ORAI-mediated calcium release-activated calcium channel, or CRAC channel, is a key source of calcium influx for intracellular Ca2+ signaling and effector function in T lymphocytes. STIM in the ER membrane senses depletion of ER calcium stores and moves to ER-plasma membrane (PM) junctions where it recruits PM ORAI channels and triggers store-operated Ca2+ entry. We have shown previously that septins 4 and 5 are crucial for efficient STIM1-ORAI1 cluster formation following store depletion. Septins were previously reported to specify PM barriers at certain sites and to serve as scaffolds that recruit signaling proteins, but their detailed role in calcium signaling remained elusive. We used live-cell super-resolution microscopy and single-molecule tracking to map ORAI1, STIM1, ER-PM junctions, and membrane-localized septin 4 in resting cells and store-depleted cells. We further investigated the trajectories of ORAI1 channels in septin 4/5-deficient cells or in cells where the interaction between ORAI1 and STIM1 was abolished by mutagenesis. Our data show that septins 4/5 are needed to maintain a normal complement of functional ER-PM junctions, and to support efficient STIM-ORAI interaction at the junctions. Septins neither promote ORAI localization to junctions independent of STIM, nor do they specify corrals surrounding junctions to confine ORAI, but they do contribute to decreased ORAI1 mobility across the entire cell footprint after store depletion. This additional factor could support calcium signalling by slowing the escape of ORAI1 channels from junctions after they have been recruited there by STIM. Thus live-cell single-molecule tracking has allowed us to document three primary mechanisms by which septins 4/5 enhance STIM-ORAI signaling.