Global Ca2 Research Articles

Different roles of P2X4- and P2Y1-receptors in the atrial pathogenesis during prolonged pressure overload.

Pressure overload (PO) involve increase in interstitial fluid shear in cardiac muscle during cardiac cycles due to detachment of myocytes from the extracellular matrix and myocyte slippage. However, the effects of shear stress on cardiac muscle and their pathophysiological roles are poorly understood. We have recently discovered that shear stress induces global Ca2+ waves via activation of P2 purinergic receptors in atrial myocytes by ATP released from the cells, and that this ATP release is mediated by connexin 43 (Cx43) gap junction hemichannels. In this study, we investigated role of shear stress signaling in PO-induced atrial remodeling and failure. We used a transverse aortic constriction (TAC) rat model to increase afterload and examined shear stress responses and remodeling of their signaling molecules during the development of pathological phenotypes by progressive TAC. After 5-week TAC, atrial dimension was enlarged with ventricular hypertrophy, which was followed by severe atrial dilation as well as ventricular failure at long-term (>20-week) TAC. Atrial myocytes were enlarged following 5-week TAC, which was maintained during more prolonged TAC. In the 5-week-TAC left atrial (LA) cells, ATP release was increased with no changes in Cx43 expression and in Cx43 hemichannel-mediated dye (calcein) flux. At this stage, however, function (cation current) of P2X4 receptor (P2X4R) and their protein expressions were largely enhanced. In failed LA cells, reduction in the Cx43 expression and smaller Cx43 hemichannel-mediated dye flux were observed. In failed LA myocytes, shear-induced longitudinal Ca2+ waves that were mediated by P2Y1 receptor (P2Y1R), were enhanced with higher P2Y1R expression. The opposite changes in the shear-induced responses mediated by P2X4R and P2Y1R and in their expressions suggest that P2X4R and P2Y1R may play beneficial and harmful role, respectively, in the PO-dependent atrial pathogenesis.

An integrate-and-fire approach to Ca2+ signaling. Part I: Renewal model

In computational neuroscience integrate-and-fire models capture the spike generation by a subthreshold dynamics supplemented by a simple fire-and-reset rule; they allow for a numerically efficient and analytically tractable description of stochastic single cell as well as network dynamics. Stochastic spiking is also a prominent feature of Ca2+ signaling which suggests to adopt the integrate-and-fire approach for this fundamental biophysical process. The model introduced here consists of two components describing 1) activity of clusters of inositol-trisphosphate receptor channels and 2) dynamics of the global Ca2+ concentrations in the cytosol. The cluster dynamics is given in terms of a cyclic Markov chain, capturing the puff, i.e., the punctuated release of Ca2+ from intracellular stores. The cytosolic Ca2+ concentration is described by an integrate-and-fire dynamics driven by the puff current. For the cyclic Markov chain we derive expressions for the statistics of the interpuff interval, the single-puff strength and the puff current assuming constant cytosolic Ca2+. The latter condition is often well approximated because cytosolic Ca2+ varies much slower than the cluster activity does. Furthermore, because the detailed two-component model is numerically expensive to simulate and difficult to treat analytically, we develop an analytical framework to approximate the driving puff current of the stochastic cytosolic Ca2+ dynamics by a temporally uncorrelated Gaussian noise. This approximation reduces our two-component system to an integrate-and-fire model with a nonlinear drift function and a multiplicative Gaussian white noise, a model that is known to generate a renewal spike train, i.e., a point process with statistically independent interspike intervals. The model allows for fast numerical simulations, permits to derive analytical expressions for the rate of Ca2+ spiking and the coefficient of variation of the interspike interval, and to approximate the interspike interval density and the spike train power spectrum. Comparison of these statistics to experimental data is discussed.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

Characterization of Endo-Lysosomal Cation Channels Using Calcium Imaging.

Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca2+-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca2+ measurements, (2) peri-endo-lysosomal Ca2+ imaging using genetically encoded Ca2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca2+ imaging by directing Ca2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca2+ imaging.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

CAMP-PKA signaling modulates the automaticity of human iPSC-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity, which is regulated by both Ca2+ and membrane clocks. To investigate the mechanisms coupling these clocks, we tested three hypotheses: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by local Ca2+ releases (LCRs) and cAMP/PKA-dependent coupling of Ca2+ clock to M clock; (2) the LCR period indicates the level of crosstalk within the coupled-clock system; and (3) perturbing the activity of even one clock can lead to hiPSC-CM-altered automaticity due to diminished crosstalk within the coupled-clock system. By measuring the local and global Ca2+ transients, we found that the LCRs properties are correlated with the spontaneous beat interval. Changes in cAMP-dependent coupling of the Ca2+ and M clocks, caused by a pharmacological intervention that either activates the β-adrenergic or cholinergic receptor or upregulates/downregulates PKA signaling, affected LCR properties, which in turn altered hiPSC-CMs automaticity. Clocks' uncoupling by attenuating the pacemaker current If or the sarcoplasmic reticulum Ca2+ kinetics, decreased hiPSC-CMs beating rate, and prolonged the LCR period. Finally, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar. In conclusion, hiPSC-CM automaticity is controlled by the coupled-clock system whose function is mediated by Ca2+-cAMP-PKA signaling.

Physiological levels of fluid shear stress modulate vascular function through TRPV4 sparklets.

Endothelial calcium (Ca 2+) signaling plays a major role in regulating vasodilation in response to fluid shear stress (FSS) generated by blood flow. Local Ca 2+ influx through single transient receptor potential channel subfamily V member 4 (TRPV4) (termed "sparklets") activated by low concentrations of chemical and biological stimuli has been revealed to modulate vascular function. However, the range in which FSS can initiate TRPV4 sparklets to induce vasodilation is unknown. Here, we assess the activity of TPRV4 sparklets induced by various physiological levels of FSS and investigate the mechanisms involving these Ca 2+ signals in FSS-induced vasodilation. Intact small mesenteric arteries are used for Ca 2+ imaging with a GCaMP2(TRPV4-KO) mouse model and high-speed confocal systems. Markedly increased local Ca 2+ signals are observed in the endothelium under 4-8 dyne/cm 2 FSS, whereas FSS >8 dyne/cm 2 causes global Ca 2+ influx. Further analysis shows that TRPV4 channels form a four-channel group to mediate Ca 2+ sparklets under certain levels of FSS. The large Ca 2+ influx hyperpolarizes endothelial cells by stimulating intermediate (IK)- and small (SK)-conductance Ca 2+-sensitive potassium channels, leading to hyperpolarization of the surrounding smooth muscle cells and ultimately causing endothelium-dependent vasodilation. In conclusion, Ca 2+ influx transits through a small number of endothelial TRPV4 channels opened by certain levels of FSS, which activates the Ca 2+-sensitive IK and SK channels to cause vasodilation.

KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically ‘licenses’ the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity (‘punctate’ Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete ‘diffuse’ mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

Effects of moderate-continuous and high-intensity interval aerobic training on cardiac function of spontaneously hypertensive rats.

The aim of this study was to verify the effects of moderate-intensity continuous (MICT) and high-intensity interval (HIIT) aerobic training on cardiac morphology and function and the mechanical properties of single cardiomyocytes in spontaneously hypertensive rats (SHR) in the compensated phase of hypertension. Sixteen-week-old male SHR and normotensive Wistar (WIS) rats were allocated to six groups of six animals each: SHR CONT or WIS CONT (control); SHR MICT or WIS MICT (underwent MICT, 30 min/day, five days per week for eight weeks); and SHR HIIT or WIS HIIT (underwent HIIT, 30 min/day, five days per week for eight weeks). Total exercise time until fatigue and maximum running speed were determined using a maximal running test before and after the experimental period. Systolic (SAP), diastolic (DAP), and mean (MAP) blood pressures were measured using tail plethysmography before and after the experimental period. Echocardiographic evaluations were performed at the end of the experimental period. The rats were euthanized after in vivo assessments, and left ventricular myocytes were isolated to evaluate global intracellular Ca2+ transient ([Ca2+]i) and contractile function. Cellular measurements were performed at basal temperature (~37°C) at 3, 5, and 7 Hz. The results showed that both training programs increased total exercise time until fatigue and, consequently, maximum running speed. In hypertensive rats, MICT decreased SAP, DAP, MAP, interventricular septal thickness during systole and diastole, and the contraction amplitude at 5 Hz. HIIT increased heart weight and left ventricular wall thickness during systole and diastole and reduced SAP, MAP, and the time to peak [Ca2+]i at all pacing frequencies. In conclusion, both aerobic training protocols promoted beneficial adaptations to cardiac morphology, function, and mechanical properties of single cardiomyocytes in SHR.

A localized calcium transient and polar body abscission

ABSTRACT Polar body emission is a special form of cytokinesis in oocyte meiosis that ensures the correct number of chromosomes in reproduction-competent eggs. The molecular mechanism of the last step, polar body abscission, is poorly understood. While it has been proposed that Ca2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca2+ have been difficult to document in oocyte meiosis except for the global Ca2+ wave induced by sperm at fertilization. Here, we find that microinjection of the calcium chelator dibromo-BAPTA inhibits polar body abscission in Xenopus laevis oocytes. Using a novel, microtubule-targeted ratio-metric calcium sensor, we detected a calcium transient that is focused at the contractile ring-associated plasma membrane and which occurred after anaphase and constriction of the contractile ring but prior to abscission. This calcium transient was confirmed by mobile calcium probes. Further, the Ca2+-sensitive protein kinase Cβ C2 domain transiently translocated to the contractile ring-associated membrane simultaneously with the calcium transient. Collectively, these results demonstrate that a calcium transient, apparently originating at the contractile ring-associated plasma membrane, promotes polar body abscission.

Discerning the role of IP3R1 activation by Gq/G11 signaling in murine lymphatic collecting vessel pacemaking

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure‐dependent frequency that increases with elevations in intraluminal pressure. Our recent work posits a putative role for lymphatic muscle cells (LMCs) as the pacemaker cell that regulates this pressure‐dependent phenomenon through activation of the Ca2+‐activated chloride channel anoctamin 1 (Ano1) in an IP3R1‐dependent process. However, the specific mechanisms underlying IP3R1 activation and SR Ca2+ release in lymphatic muscle cells is unknown. We tested the hypothesis that cLV pressure‐dependent pacemaking by the IP3R1‐Ano1 axis is downstream of the canonical Gq/G11‐PLC‐β signaling pathway. To this end, we crossed mice with the smooth muscle‐specific tamoxifen‐inducible Cre, MYH11CreERT2, to the fluorescent reporter ROSAmT/mG and sorted GFP+ LMCs from an isolated murine inguinal‐axillary (IngAx) lymphatic cLVs. Murine lymphatic muscle cell expression of Gq and G11 were assessed via RT‐PCR. We crossed MYH11CreERT2 mice with IP3R1fl/fl or G11KO/KO‐Gqfl/fl mice to achieve IP3R1ismKO (MYH11CreERT2‐IP3R1fl/fl) and double G11KO‐GqismKO (MYH11CreERT2‐ G11KO/KO‐Gqfl/fl) mice. We tested the function of the IngAx cLVs isolated from these mice, as well as their respective heterozygous WT/KO and fl/fl controls, using isolated vessel isobaric myography. Ca2+ imaging using IP3R1ismKO‐GCaMP6f mice, and membrane potential recordings using sharp electrodes. IngAx cLVs from G11KO‐GqismKO mice displayed a significant reduction in contraction frequency and vessel tone, however a pressure‐dependent increase in frequency was observed. Similarly, IngAx cLVs isolated from IP3R1ismKO mice had significantly lower vessel tone, a significant reduction in contraction frequency at all pressures tested, and a significantly higher contraction amplitude. Critically, contraction frequency in IngAx cLVs isolated from IP3R1ismKO mice did not increase with pressure in contrast to IngAx cLVs isolated from G11KO‐GqismKO mice and wildtype mice. In wildtype (WT) MYH11CreERT2‐GCaMP6f controls, Ca2+ puffs and/or Ca2+ waves were observed in the majority of LMCs during the diastolic period, intervening the bright global Ca2+ flashes indicative of Ca2+ entry through L‐type Ca2+ channels during the LMC action potential (AP). These local diastolic Ca2+ transients (CaTs) persisted in the presence of nifedipine, and the frequency and spatial spread of CaTs increased with pressure. Diastolic CaTs were largely absent in LMCs of IP3R1ismKO mice, and their diastolic depolarization recapitulated the lower rate observed in previous studies of Ano1smKO cLVs. However, APs recorded in IP3R1ismKO significantly increased AP plateau duration that corresponds with an increased Ca2+ flash duration. We conclude that the Gq/G11 dependent signaling is involved in pressure‐dependent pacemaking in murine cLVs, particularly at low pressure, but their deletion is insufficient to reproduce the IP3R1ismKO phenotype.

Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart.We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/−) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/− myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A.Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

P2X4 and P2X7 are essential players in basal T cell activity and Ca2+ signaling milliseconds after T cell activation.

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca2+ microdomains. Purinergic signaling is known to be involved in Ca2+ influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca2+ live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca2+ signals but also promote initial Ca2+ microdomains tens of milliseconds after T cell stimulation. These Ca2+ microdomains were significantly decreased in T cells from P2rx4−/− and P2rx7−/− mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1–dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca2+ microdomains already in the first second of T cell activation.

Activation of cardiac connexin 43 hemichannels by shear stress and subsequent contribution of pannexins in left-side cardiac myocytes

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger global Ca2+ waves in atrial myocytes through autocrine activation of P2 receptors by connexin43 (Cx43)-mediated ATP release. We examined whether shear stress activates Cx43 hemichannels and pannexins in atrial and ventricular myocytes from right and left side of heart chambers. The activities of these hemichannels were measured as an efflux of large fluorescence dye (calcein-red/orange) to permeate these channels using two-dimensional confocal imaging method in single isolated myocytes of murine hearts.

Modulation of global Ca2+ handling by SR-Ca2+ release depending on IP3

In the heart muscle, Ca2+-induced Ca2+ release (CICR) is the main mechanism for Ca2+ increase required for the excitation-contraction coupling. However, a second mechanism for Ca2+ release from the sarcoplasmic reticulum induced by inositol 1,4,5-trisphosphate (IP3), called inositol IP3-induced intracellular Ca2+ release (IP3ICR), has been verified in cardiomyocytes. Occurrence of global Ca2+ transients as well as spontaneous Ca2+ waves (SCaW) are driven by CICR. Under pathophysiological remodeling conditions in which an up-regulation of IP3 receptors (IP3Rs) was observed, a modulatory role of IP3ICR on CICR seems plausible.

Nanoscale Organization, Regulation, and Dynamic Reorganization of Cardiac Calcium Channels.

The architectural specializations and targeted delivery pathways of cardiomyocytes ensure that L-type Ca2+ channels (CaV1.2) are concentrated on the t-tubule sarcolemma within nanometers of their intracellular partners the type 2 ryanodine receptors (RyR2) which cluster on the junctional sarcoplasmic reticulum (jSR). The organization and distribution of these two groups of cardiac calcium channel clusters critically underlies the uniform contraction of the myocardium. Ca2+ signaling between these two sets of adjacent clusters produces Ca2+ sparks that in health, cannot escalate into Ca2+ waves because there is sufficient separation of adjacent clusters so that the release of Ca2+ from one RyR2 cluster or supercluster, cannot activate and sustain the release of Ca2+ from neighboring clusters. Instead, thousands of these Ca2+ release units (CRUs) generate near simultaneous Ca2+ sparks across every cardiomyocyte during the action potential when calcium induced calcium release from RyR2 is stimulated by depolarization induced Ca2+ influx through voltage dependent CaV1.2 channel clusters. These sparks summate to generate a global Ca2+ transient that activates the myofilaments and thus the electrical signal of the action potential is transduced into a functional output, myocardial contraction. To generate more, or less contractile force to match the hemodynamic and metabolic demands of the body, the heart responds to β-adrenergic signaling by altering activity of calcium channels to tune excitation-contraction coupling accordingly. Recent accumulating evidence suggests that this tuning process also involves altered expression, and dynamic reorganization of CaV1.2 and RyR2 channels on their respective membranes to control the amplitude of Ca2+ entry, SR Ca2+ release and myocardial function. In heart failure and aging, altered distribution and reorganization of these key Ca2+ signaling proteins occurs alongside architectural remodeling and is thought to contribute to impaired contractile function. In the present review we discuss these latest developments, their implications, and future questions to be addressed.

Sphingosine-1-phosphate induces Ca<sup>2+</sup> mobilization via TRPC6 channels in SH-SY5Y cells and hippocampal neurons

Sphingosine-1-phosphate (S1P) is a widely expressed biologically active sphingolipid that plays an important role in cell differentiation, migration, proliferation, metabolism and apoptosis. S1P activates various signaling pathways, some of which evoke Ca<sup>2+</sup> signals in the cytosol. Few studies have focused on the mechanism by which S1P evokes Ca<sup>2+</sup> signals in neurons. Here, we show that S1P evokes global Ca<sup>2+</sup> signals in SH-SY5Y cells and hippocampal neurons. Removal of extracellular calcium largely abolished the S1P-induced increase in intracellular Ca<sup>2+</sup>, suggesting that the influx of extracellular Ca<sup>2+</sup> is the major contributor to this process. Moreover, we found that S1P-induced Ca<sup>2+</sup> mobilization is independent of G protein-coupled S1P receptors. The TRPC6 inhibitor SAR7334 suppressed S1P-induced calcium signals, indicating that the TRPC6 channel acts as the downstream effector of S1P. Using patch-clamp recording, we showed that S1P activates TRPC6 currents. Two Src tyrosine kinase inhibitors, Src-I1 and PP2, dramatically inhibited the activation of TRPC6 by S1P. Taken together, our data suggest that S1P activates TRPC6 channels in a Src-dependent way to induce Ca<sup>2+</sup> mobilization in SH-SY5Y cells and hippocampal neurons.

Acidic Ca2+ stores and immune-cell function

Acidic organelles act as intracellular Ca2+ stores; they actively sequester Ca2+ in their lumina and release it to the cytosol upon activation of endo-lysosomal Ca2+ channels. Recent data suggest important roles of endo-lysosomal Ca2+ channels, the Two-Pore Channels (TPCs) and the TRPML channels (mucolipins), in different aspects of immune-cell function, particularly impacting membrane trafficking, vesicle fusion/fission and secretion. Remarkably, different channels on the same acidic vesicles can couple to different downstream physiology. Endo-lysosomal Ca2+ stores can act under different modalities, be they acting alone (via local Ca2+ nanodomains around TPCs/TRPMLs) or in conjunction with the ER Ca2+ store (to either promote or suppress global ER Ca2+ release). These different modalities impinge upon functions as broad as phagocytosis, cell-killing, anaphylaxis, immune memory, thrombostasis, and chemotaxis.

Distinct dendritic Ca2+ spike forms produce opposing input-output transformations in rat CA3 pyramidal cells.

Proper integration of different inputs targeting the dendritic tree of CA3 pyramidal cells (CA3PCs) is critical for associative learning and recall. Dendritic Ca2+ spikes have been proposed to perform associative computations in other PC types by detecting conjunctive activation of different afferent input pathways, initiating afterdepolarization (ADP), and triggering burst firing. Implementation of such operations fundamentally depends on the actual biophysical properties of dendritic Ca2+ spikes; yet little is known about these properties in dendrites of CA3PCs. Using dendritic patch-clamp recordings and two-photon Ca2+ imaging in acute slices from male rats, we report that, unlike CA1PCs, distal apical trunk dendrites of CA3PCs exhibit distinct forms of dendritic Ca2+ spikes. Besides ADP-type global Ca2+ spikes, a majority of dendrites expresses a novel, fast Ca2+ spike type that is initiated locally without bAPs, can recruit additional Na+ currents, and is compartmentalized to the activated dendritic subtree. Occurrence of the different Ca2+ spike types correlates with dendritic structure, indicating morpho-functional heterogeneity among CA3PCs. Importantly, ADPs and dendritically initiated spikes produce opposing somatic output: bursts versus strictly single-action potentials, respectively. The uncovered variability of dendritic Ca2+ spikes may underlie heterogeneous input-output transformation and bursting properties of CA3PCs, and might specifically contribute to key associative and non-associative computations performed by the CA3 network.

Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction—NAADP to NAADPH—is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.