Capacitative Ca2 Research Articles

Magnetic fields promote a pro-survival non-capacitative Ca2+ entry via phospholipase C signaling

The ability of magnetic fields (MFs) to promote/increase Ca2+ influx into cells is widely recognized, but the underlying mechanisms remain obscure. Here we analyze how static MFs of 6mT modulates thapsigargin-induced Ca2+ movements in non-excitable U937 monocytes, and how this relates to the anti-apoptotic effect of MFs. Magnetic fields do not affect thapsigargin-induced Ca2+ mobilization from endoplasmic reticulum, but significantly increase the resulting Ca2+ influx; this increase requires intracellular signal transduction actors including G protein, phospholipase C, diacylglycerol lipase and nitric oxide synthase, and behaves as a non-capacitative Ca2+ entry (NCCE), a type of influx with an inherent signaling function, rather than a capacitative Ca2+ entry (CCE). All treatments abrogating the extra Ca2+ influx also abrogate the anti-apoptotic effect of MFs, demonstrating that MF-induced NCCE elicits an anti-apoptotic survival pathway.

Hypoxic remodelling of Ca 2+ stores does not alter human cardiac myofibroblast invasion

Cardiac fibroblasts are the most abundant cell type in the heart, and play a key role in the maintenance and repair of the myocardium following damage such as myocardial infarction by transforming into a cardiac myofibroblast (CMF) phenotype. Repair occurs through controlled proliferation and migration, which are Ca 2+ dependent processes, and often requires the cells to operate within a hypoxic environment. Angiotensin converting enzyme (ACE) inhibitors reduce infarct size through the promotion of bradykinin (BK) stability. Although CMF express BK receptors, their activity under the reduced O 2 conditions that occur following infarct are entirely unexplored. Using Fura-2 microfluorimetry on primary human CMF, we found that hypoxia significantly increased the mobilisation of Ca 2+ from intracellular stores in response to BK whilst capacitative Ca 2+ entry (CCE) remained unchanged. The enhanced store mobilisation was due to a striking increase in CMF intracellular Ca 2+-store content under hypoxic conditions. However, BK-induced CMF migration or proliferation was not affected following hypoxic exposure, suggesting that Ca 2+ influx rather than mobilisation is of primary importance in CMF migration and proliferation.

Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non‐cystic fibrosis epithelia

The lantibiotic duramycin (Moli1901, Lancovutide) has been suggested as a drug of choice in the treatment for cystic fibrosis (CF). It has been proposed that duramycin may stimulate chloride secretion through Ca²(+) -activated Cl⁻ channels (CaCC). We investigated whether duramycin exhibited any effect on Cl⁻ efflux and intracellular Ca²(+) concentration ([Ca²(+)](i)) in CF and non-CF epithelial cells. Duramycin did stimulate Cl⁻ efflux from CF bronchial epithelial cells (CFBE) in a narrow concentration range (around 1 μM). However, 100 and 250 μM of duramycin inhibited Cl⁻ efflux from CFBE cells. An inhibitor of the CF transmembrane conductance regulator (CFTR(inh)₋₁₇₂) and a blocker of the capacitative Ca²(+) entry, gadolinium chloride, inhibited the duramycin-induced Cl⁻ efflux. No effect on Cl⁻ efflux was observed in non-CF human bronchial epithelial cells (16HBE), human airway submucosal gland cell line, human pancreatic epithelial cells, CF airway submucosal gland epithelial cells, and CF pancreatic cells. The [Ca²(+)](i) was increased by 3 μM duramycin in 16HBE cells, but decreased after 1, and 3 μM of duramycin in CFBE cells. The results suggest that the mechanism responsible for the stimulation of Cl⁻ efflux by duramycin is mainly related to unspecific changes of the cell membrane or its components rather than to effects on CaCC.

Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca(2+) entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca(2+) entry independent of intracellular Ca(2+) stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca(2+) signaling via the CRAC channel.

Features of Ca2+ signaling in proliferating and in differentiating murine myoblasts

The state of the Ca2+ signaling system has been assessed in proliferating and in differentiating C2C12 myoblasts. Proliferating myoblasts exhibit no features of a functional system: the intracellular ATP-controlled Ca2+ store is low (perhaps only mitochondrial) and no Ca2+ entry from the medium is registered upon its exhaustion, there is no cytosolic response to KCl-induced depolarization. The Ca2+ signaling system starts to form at the early stages of differentiation (within 10 h after transfer of cells to the differentiation medium). This is seen as appearance of capacitive and voltage-dependent Ca2+ entry and its accumulation in the endoplasmic reticulum. A small contribution to the ATP-evoked rise in cytosolic Ca2+ is also made by mitochondria.

Stretch-Induced TRPC4 Downregulation is Accompanied By Reduced Capacitative Ca2+ Entry in WKY But Not SHR Mesenteric Smooth Muscle Cells

Our previous work showed that a 6-hour cyclic stretch significantly decreases TRPC4 protein expression and capacitative Ca2+ entry in vascular smooth muscle cells from Sprague-Dawley rats. To parallel these studies, mesenteric smooth muscle cells from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar Kyoto (WKY) rats, were subjected to stretch. TRPC4 protein expression was evaluated by Western blot and Ca2+ mobilization was measured using fura-2. As in Sprague-Dawley cells, a 6-hour stretch resulted in a significant down-regulation of TRPC4 protein in both SHR and WKY mesenteric smooth muscle cells. While WKY cells showed a stretch-induced decrease in Ca2+ dynamics to accompany the reduction in TRPC4 expression, mesenteric smooth muscle cells from SHR showed a stretch-induced increase in both the release of stored Ca2+ and capacitative Ca2+ entry. TRPC4 proteins may be working as store-operated channels in normotensive vascular smooth muscle cells and their down-regulation by stretch may be a protective mechanism to prevent additional Ca2+ influx during stretch. The stretch-induced increase in capacitative Ca2+ entry in SHR may be due to a compensatory upregulation of non-TRPC4 channels or an increase in store-operated signaling or channel activity.

Expression of Orai genes and ICRAC activation in the human retinal pigment epithelium

The retinal pigment epithelium (RPE) fulfills a large variety of tasks that are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion, or transepithelial ion transport, are regulated by increases in intracellular Ca²(+) as second-messenger. Despite the multitude of Ca²(+)-dependently regulated functions, only few Ca²(+) channels have been described so far in the RPE to couple Ca²(+) conductance and Ca²(+) signaling. RT-PCR experiments with mRNA of freshly isolated RPE cells as well as from the RPE cell line ARPE-19 and measurements of the intracellular free Ca²(+) concentration were performed. The RT-PCR experiments revealed the expression of the I(CRAC) channel proteins Orai 1, 2, and 3 and their stimulators Stim-1 and Stim-2. The classic maneuver to stimulate capacitive Ca²(+) entry (depletion of Ca²(+) stores by 1 μM thapsigargin under extracellular Ca²(+)-free conditions and then re-adding extracellular Ca²(+)) led to an increase in intracellular free Ca²(+), which could be blocked by application of a high concentration of 2-APB (75 μM) either before or during induction of capacitive Ca²(+) entry. On the other hand, application of a low concentration of 2-APB (2 μM) led to enhancement of the Ca²(+) increase induced by capacitive Ca²(+) entry. Depletion of cytosolic Ca²(+) stores by administration of an extracellular divalent cation-free solution led to an increase in the whole-cell conductance. With these data we show a new Ca²(+) entry pathway linked to the Ca²(+)/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists. The expression of Orai channels enables the RPE cells to generate sustained or repetitive Ca²(+) signals as they are known to be induced by different stimuli like ATP, bFGF, and the stimulation with photoreceptor outer segments.

15-HETE mediates sub-acute hypoxia-induced TRPC1 expression and enhanced capacitative calcium entry in rat distal pulmonary arterial myocytes

Sub-acute hypoxia causes pulmonary vasoconstriction (HPV) is associated with increased intracellular Ca 2+ concentration ([Ca 2+] i) and contraction of pulmonary arterial smooth muscle cells (PASMCs). We previous have demonstrated that 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of arachidonic acid by 15-lipoxygenase (15-LO), causes elevated [Ca 2+] i in PASMCs partly through Ca 2+ entry via other than L-type Ca 2+ channels. In this study, we used SKF96365/La 3+ (SOCC antagonists) and Nordihydro-guiairetic acid (NDGA, a blockage of 15-LO) to examine the effect of 15-HETE on capacitative Ca 2+ entry and activity/expression of store-operated Ca 2+ channels (SOCCs) during sub-acute hypoxic procedure and the contribution of SOCCs on the maintenance of vascular tones. The results showed that the 15-HETE induced constriction of PA rings from normoxic and sub-acute hypoxic rats can be abolished by SKF96365 and La 3+. Capacitative Ca 2+ entry (CCE) was also enhanced in PASMCs cultured with 15-HETE under sub-acute hypoxic condition (3% O 2, 48 h) and incubation with NDGA in PASMCs can greatly suppress this enhancement. Moreover, TRPC1, not TRPC4 and TRPC6, mRNA and protein expression were increased in PASMCs during these procedures. Meanwhile, the effect of 15-HETE on CCE and TRPC1 expression under sub-acute hypoxic cultivation were greatly suppressed in 15-LO knockdown PASMCs and PAs. These results suggest that 15-HETE mediated HPV through increased TRPC1 expression, leading to enhanced CCE, contributing to the maintenance of vascular tone.

Up-regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer's disease

The cellular pathology of Alzheimer's disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca2+ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non-transgenic neurons. Furthermore, despite increased intracellular Ca2+ release in response to ryanodine, we found that basal Ca2+, K+-evoked Ca2+ responses, and capacitative Ca2+ entry were no different in TgCRND8 neurons compared with non-transgenic neurons. Therefore, as RyR3 up-regulation did not affect neuronal health or global Ca2+ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non-transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer's disease.

Mechanism of carteolol-induced cytosolic Ca2+ mobilization in cultured vascular smooth muscle cells

An increase in cytosolic calcium concentration triggers intracellular signal transduction in vascular cells, which then regulates the vascular contraction. In the present study, the regulatory mechanism of carteolol on the intracellular free Ca2+ ([Ca2+]i) mobilization was investigated in cultured A7r5 vascular smooth muscle cells. The A7r5 cells were cultured and loaded with fura-2-AM, which was used as a Ca2+ sensitive fluorescent probe. In both the presence and absence of external Ca2+, carteolol increased [Ca2+]i with a dose-dependent manner in A7r5 cells at concentrations between 608µM and 6.08µM. In a Ca2+-containing buffer, carteolol-induced [Ca2+]i showed an initial peak followed by a secondary and persistent plateau. Pretreatment of the cells with La3+, the plasma membrane Ca2+ pump inhibitor, and nifedipine, a L-type Ca2+ channel inhibitor, both partially restrained the carteolol-induced initial peak in [Ca2+]i by 92% and 86%, respectively. Pretreatment of the cells with adrenoceptor antagonists, prazosin inhibited the [Ca2+]i response by 80%, and propranolol enhanced the response by 61%. In the Ca2+−-free buffer, pretreatment of the cells with carteolol inhibited the endoplasmic reticulum Ca2+ pump inhibitor of thapsigargin-induced [Ca2+]i increase by 97%. Pretreatment of the cells with thapsigargin also inhibited the carteolol-induced [Ca2+]i rise by 98%. The internal Ca2+ release induced by the carteolol was partially inhibited by U73122 (phospholipase C inhibitor) and aristolochic acid, quinacrine (phospholipase A2 inhibitors). After incubation of carteolol in the Ca2+-free buffer, the addition of CaCl2 increased the Ca2+ influx, implying that the release of Ca2+ from internal stores further induced capacitative Ca2+ entry. These results suggest that carteolol-induced [Ca2+]i increase is mediated by the initial influx via the α1-adrenoceptor, L-type Ca2+ channel, nonselective calcium entry channels and release of Ca2+ from an intracellular store, which is mainly in the endoplasmic reticulum followed by capacitative Ca2+ entry but decrease via the β2-adrenoceptor. The intracellular Ca2+ release was also modulated by phospholipase A2, C-coupled events.

Ca2+ Hot Spots on the Mitochondrial Surface Are Generated by Ca2+ Mobilization from Stores, but Not by Activation of Store-Operated Ca2+ Channels

Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca(2+) during stimulation because of their vicinity to microdomains of high [Ca(2+)], the direct proof of Ca(2+) hot spots' existence is still lacking. Thanks to a GFP-based Ca(2+) probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca(2+) mobilization, the [Ca(2+)] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca(2+)] to which mitochondria are exposed during capacitative Ca(2+) influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca(2+) channels. These results demonstrate that mitochondria are exposed to Ca(2+) hot spots close to the ER but are excluded from the regions where capacitative Ca(2+) influx occurs.

Possible involvement of Ca2+ entry and its pharmacological characteristics responsible for endothelium-dependent, NO-mediated relaxation induced by thapsigargin in guinea-pig aorta.

Thapsigargin, a specific inhibitor of Ca(2+)-pump Ca(2+)-ATPase in the sarcoplasmic/endoplasmic reticulum (SR/ER), produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features of thapsigargin-induced endothelium-dependent relaxation were functionally characterized in the isolated guinea-pig aorta especially focusing on the Ca2+ mobilization mechanisms in endothelial cells. Thapsigargin-induced endothelium-dependent vascular relaxation was markedly suppressed by N(G)-nitro-L-arginine (L-NNA) and calmidazolium, suggesting that the vascular relaxation to thapsigargin is largely attributable to endothelium-derived nitric oxide (NO) produced as a result of the activation of Ca2+, calmodulin-dependent NO synthase (NOS). Removal of Ca2+ from the external solution abolished the endothelium-dependent relaxation of guinea-pig aorta in response to thapsigargin. Thapsigargin-induced endothelium-dependent relaxation was inhibited more strongly compared with the endothelium-independent relaxation to an NO donor, SIN-1 (3-(4-morpholinyl)-sydnonimine), when the artery preparation was preconstricted with a high concentration (80 mM) of KCl instead of agonistic stimulation. Endothelium-dependent relaxation induced by thapsigargin was not affected by diltiazem, a blocker of L-type voltage-gated Ca2+ channels. SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole) and Ni2+, both of which block capacitative Ca(2+) entry, did not show any appreciable inhibitory effects on the endothelium-dependent relaxation to thapsigargin. These findings suggest that in guinea-pig aorta, endothelium-dependent NO-mediated relaxation induced by thapsigargin is preceded by the increase in the cytosolic free Ca2+ concentrations ([Ca2+]cyt) following the depletion of stored Ca2+ in thapsigargin-sensitive store sites in endothelial cells. Although the increase in [Ca2+]cyt responsible for the activation of endothelium NOS leading to thapsigargin-induced vascular relaxation may be ascribed to the capacitative Ca2+ entry from extracellular space, the Ca2+ entry mechanism stimulated with thapsigargin is deficient in sensitivity to SK&F96365 and Ni2+ in the endothelium of guinea-pig aorta.

Orai1 and STIM1 Mediate Capacitative Ca2+ Entry in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 and STIM1 mediates the sustained component of capacitative Ca2+ entry (CCE) but the molecular candidate(s) that mediate the transient component of CCE remains unknown. The aim of the present study was to further examine if Orai1 mediates the transient component of CCE through activation of STIM1 protein in mouse PASMCs.In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i).The transient but not the sustained rise in [Ca2+]i was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 siRNA. Moreover, over-expression of STIM1 enhanced the rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to co-immunoprecipitate with STIM1 and immunostaining showed co-localization of Orai1 and STIM1 proteins. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

A timely characterization of vasopressin-sensitive adenylyl cyclase isoforms in the mouse inner medullary collecting duct

second messenger signaling in the kidney mediates several of the organ's basic physiological functions including the regulation of blood pressure, control of fluid volume, and maintenance of the body's water homeostasis in times of duress. Tight regulation of the second messenger cAMP is critical

Up‐regulation of the type 3 ryanodine receptor is neuroprotective in the TgCRND8 mouse model of Alzheimer’s disease

The cellular pathology of Alzheimer's disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca2+ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non-transgenic neurons. Furthermore, despite increased intracellular Ca2+ release in response to ryanodine, we found that basal Ca2+, K+-evoked Ca2+ responses, and capacitative Ca2+ entry were no different in TgCRND8 neurons compared with non-transgenic neurons. Therefore, as RyR3 up-regulation did not affect neuronal health or global Ca2+ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non-transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer's disease.

Arachidonic acid stimulates extracellular Ca2+ entry in rat pancreatic β cells via activation of the noncapacitative arachidonate-regulated Ca2+ (ARC) channels

Arachidonic acid (AA) is generated in the pancreatic islets during glucose stimulation. We investigated whether AA activated extracellular Ca2+ entry in rat pancreatic β cells via a pathway that was independent of the activation of voltage-gated Ca2+ channels. The AA triggered [Ca2+]i rise did not involve activation of GPR40 receptors or AA metabolism. When cells were voltage clamped at −70mV, the AA-mediated intracellular Ca2+ release was accompanied by extracellular Ca2+ entry. AA accelerated the rate of Mn2+ quench of indo-1 fluorescence (near the Ca2+-independent wavelength of indo-1), reflecting the activation of a Ca2+-permeable pathway. The AA-mediated acceleration of Mn2+ quench was inhibited by La3+ but not by 2-APB (a blocker of capacitative Ca2+ entry), suggesting the involvement of arachidonate-regulated Ca2+ (ARC) channels. Consistent with this, intracellular application of the charged membrane-impermeant analog of AA, arachidonyl-coenzyme A (ACoA) triggered extracellular Ca2+ entry, as well as the activation of a La3+-sensitive small inward current (1.7pA/pF) at −70mV. Our results indicate that the activation of ARC channels by intracellular AA triggers extracellular Ca2+ entry. This action may contribute to the effects of AA on Ca2+ signals and insulin secretion in rat β cells.

Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase

Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.

α‐Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH‐SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

STIM2 Regulates Capacitive Ca 2+ Entry in Neurons and Plays a Key Role in Hypoxic Neuronal Cell Death

Excessive cytosolic calcium ion (Ca(2+)) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca(2+) entry (CCE) is a process whereby depletion of intracellular Ca(2+) stores causes the activation of plasma membrane Ca(2+) channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)-resident Ca(2+) sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca(2+) concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca(2+) accumulation in neurons. Neurons from Stim2(-/-) mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2(-/-) mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.