Time-resolved role of P2X4 and P2X7 during CD8+ T cell activation.

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.

T cell Ca2+ microdomains through the lens of computational modeling.

Cellular Ca2+ signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.

Pharmacological inhibition of TRPV2 attenuates phagocytosis and lipopolysaccharide-induced migration of primary macrophages.

BACKGROUND AND PURPOSE: In macrophages, transient receptor potential vanilloid 2 (TRPV2) channel contributes to various cellular processes such as cytokine production, differentiation, phagocytosis and migration. Due to a lack of selective pharmacological tools, its function in immunological processes is not well understood and the identification of novel and selective TRPV2 modulators is highly desirable. EXPERIMENTAL APPROACH: Novel and selective TRPV2 modulators were identified by screening a compound library using Ca2+ influx assays with human embryonic kidney 293 (HEK293) cells heterologously expressing rat TRPV2. Hits were further characterized and validated with Ca2+ influx and electrophysiological assays. Phagocytosis and migration of macrophages were analysed and the contribution of TRPV2 to the generation of Ca2+ microdomains was studied by total internal reflection fluorescence microscopy (TIRFM). KEY RESULTS: The compound IV2-1, a dithiolane derivative (1,3-dithiolan-2-ylidene)-4-methyl-5-phenylpentan-2-one), is a potent inhibitor of heterologously expressed TRPV2 channels (IC50 = 6.3 ± 0.7 µM) but does not modify TRPV1, TRPV3 or TRPV4 channels. IV2-1 also inhibits TRPV2-mediated Ca2+ influx in macrophages. IV2-1 inhibits macrophage phagocytosis along with valdecoxib and after siRNA-mediated knockdown. Moreover, TRPV2 inhibition inhibits lipopolysaccharide-induced migration of macrophages whereas TRPV2 activation promotes migration. After activation, TRPV2 shapes Ca2+ microdomains predominantly at the margin of macrophages, which are important cellular regions to promote phagocytosis and migration. CONCLUSIONS AND IMPLICATIONS: IV2-1 is a novel TRPV2-selective blocker and underline the role of TRPV2 in macrophage-mediated phagocytosis and migration. Furthermore, we provide evidence that TRPV2 activation generates Ca2+ microdomains, which may be involved in phagocytosis and migration of macrophages.

DARTS: an open-source Python pipeline for Ca2+ microdomain analysis in live cell imaging data.

Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatio-temporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. Code available at: 10.5281/zenodo.10459243.

NAADP Signaling: New Kids on the Block.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a universal Ca2+ mobilizing second messenger essential for initiation of Ca2+ signaling. Recently, novel molecular mechanisms of both its rapid formation upon receptor stimulation and its mode of action were discovered. Dual NADPH oxidase 2 (DUOX2) and hematological and neurological expressed 1-like protein (HN1L)/Jupiter microtubule-associated homolog 2 (JPT2) were discovered as NAADP-forming enzyme and NAADP receptor/binding protein-the new kids on the block. These novel aspects are reviewed and integrated into the previous view of NAADP signaling.

Theoretical Study of the Function of the IP3 Receptor/BK Channel Complex in a Single Neuron / Estudio Teórico de la Función del Complejo Receptor-IP3/Canales BK en Neurona Única

ABSTRACT Large conductance calcium-activated potassium (BK) channels carry out many functions in the central nervous system. These channels open in response to increased cytosolic calcium ([Ca2+]cyt) concentration. The influx of calcium ions to the cytosol can occur through voltage-gated calcium channels (VGCCs) on the plasma membrane and/ or through IP3 receptors (IP3-Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum membrane. The BK channel/IP3-R/RyR interaction has been widely reported in smooth muscle but scarcely investigated in relation to neurons. The aim of this study was to theoretically explore the function of the BK/IP3-R complex by means of a computational model of a neuron that replicates the interaction between the release of Ca2+ from the endoplasmic reticulum (through IP3-Rs) and the opening of the BK channels. The mathematical models are based on the Hodgkin-Huxley formalism and the Goldbeter model. These models were implemented on Visual Basic® and differential equations were solved numerically. Distinct conditions were contemplated for BK conductance and the efflux of endoplasmic Ca2+ to the cytosol. An abrupt rise in [Ca2+]cyt (≥ 5 μM) and short duration (spark) was found to activate BK channels and either pause or stop the action potential train.

RESUMEN Los canales de potasio activados por calcio de gran conductancia (canales BK) cumplen múltiples funciones en el sistema nervioso central. Estos canales se abren en respuesta al incremento de la concentración de calcio citosólico ([Ca2+]cyt). La entrada de Ca2+ puede ocurrir a través de canales de calcio dependientes de voltaje (VGCCs) localizados en la membrana plasmática y por eflujo de Ca2+ del retículo endoplásmico (ER) causado por 1,4,5-Trifosfato (IP3) o rianodina (RyR). La interacción BK/IP3/RyR ha sido ampliamente estudiada en músculo liso, pero escasamente en neuronas. El objetivo de este estudio fue explorar teóricamente la función del complejo BK/IP3-R mediante un modelo computacional de una neurona que replica la interacción entre la liberación de Ca2+ del retículo endoplásmico (a través de IP3-Rs) y la apertura de los canales BK. Los modelos matemáticos se basan en el formalismo de Hodgkin-Huxley y el modelo de Goldbeter. Estos modelos fueron implementados en Visual Basic® y las ecuaciones diferenciales fueron resueltas por métodos numéricos. Se contemplaron distintas condiciones para la conductancia del canal BK y la salida de Ca2+ endoplásmico al citosol. Los resultados muestran que un incremento abrupto de [Ca2+] cyt (≥ 5 μM) y de corta duración (spark) activa los canales BK y producen una pausa o detiene el tren de potenciales de acción.

Ca2+ release via InsP3Rs enhances RyR recruitment during Ca2+ transients by increasing dyadic [Ca2+] in cardiomyocytes.

Excitation-contraction coupling (ECC) relies on temporally synchronized sarcoplasmic reticulum (SR) Ca2+ release via ryanodine receptors (RyRs) at dyadic membrane compartments. Neurohormones, such as endothelin-1 (ET-1), that act via Gαq-associated G protein-coupled receptors (GPCRs) modulate Ca2+ dynamics during ECC and induce SR Ca2+ release events involving Ca2+ release via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). How the relatively modest Ca2+ release via InsP3Rs elicits this action is not resolved. Here, we investigated whether the actions of InsP3Rs on Ca2+ handling during ECC were mediated by a direct influence on dyadic Ca2+ levels and whether this mechanism contributes to the effects of ET-1. Using a dyad-targeted genetically encoded Ca2+ reporter, we found that InsP3R activation augmented dyadic Ca2+ fluxes during Ca2+ transients and increased Ca2+ sparks. RyRs were required for these effects. These data provide the first direct demonstration of GPCR and InsP3 effects on dyadic Ca2+, and support the notion that Ca2+ release via InsP3Rs influences Ca2+ transients during ECC by facilitating the activation and recruitment of proximal RyRs. We propose that this mechanism contributes to neurohormonal modulation of cardiac function. This article has an associated First Person interview with the first author of the paper.

Ca2+ Microdomains, Calcineurin and the Regulation of Gene Transcription.

Ca2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca2+/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression.

Mechanisms and functions of calcium microdomains produced by ORAI channels, d-myo-inositol 1,4,5-trisphosphate receptors, or ryanodine receptors.

With the discovery of local Ca2+ signals in the 1990s the concept of 'elementary Ca2+ signals' and 'fundamental Ca2+ signals' was developed. While 'elementary Ca2+signals' relate to optical signals gained by activity of small clusters of Ca2+channels, 'fundamental signals' describe such optical signals that arise from opening of single Ca2+channels. In this review, we discuss (i) concepts of local Ca2+ signals and Ca2+ microdomains, (ii) molecular mechanisms underlying Ca2+ microdomains, (iii) functions of Ca2+ microdomains, and (iv) mathematical modelling of Ca2+ microdomains. We focus on Ca2+ microdomains produced by ORAI channels, D-myo-inositol 1,4,5-trisphosphate receptors, or ryanodine receptors. In summary, research on local Ca2+ signals in different cell models aims to better understand how cells use the Ca2+ toolkit to produce Ca2+ microdomains as relevant signals for specific cellular responses, but also how local Ca2+ signals as building blocks merge into global Ca2+ signaling.

Extra Virgin Olive Oil Phenols Vasodilate Rat MesentericResistance Artery via Phospholipase C (PLC)-CalciumMicrodomains-Potassium Channels (BKCa) Signals.

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.

Loose coupling between SK and P/Q-type Ca2+ channels in cartwheel cells of the dorsal cochlear nucleus.

Small-conductance Ca2+-activated K+ (SK) and large-conductance voltage- and Ca2+-activated K+ (BK) channels are Ca2+-activated K+ channels that control action potential firing in diverse neurons in the brain. In cartwheel cells of the dorsal cochlear nucleus, blockade of either channel type leads to excessive production of spike bursts. In the same cells, P/Q-type Ca2+ channels in plasma membrane and ryanodine receptors in endoplasmic reticulum supply Ca2+ to BK channels through Ca2+ nanodomain signaling. In this study, voltage-clamp experiments were performed in cartwheel cells in mouse brain slices to examine the Ca2+ signaling pathways underlying activation of SK channels. As with BK channels, SK channels required the activity of P/Q-type Ca2+ channels. However, this signaling occurred across Ca2+ micro- rather than nanodomain distances and was independent of Ca2+ release from endoplasmic reticulum. These differential modes of activation may lead to distinct time courses of the two K+ currents and therefore control excitability of auditory neurons across different timescales.NEW & NOTEWORTHY This study has shown for the first time that in cartwheel cells of the dorsal cochlear nucleus, small-conductance Ca2+-activated K+ (SK) channels were triggered by the activation of P/Q-type Ca2+ channels in which SK-P/Q-type coupling is mediated within the Ca2+ microdomains (loose coupling). Although Ca2+-induced Ca2+ release is able to activate large-conductance voltage- and Ca2+-activated K+ (BK) channels in cartwheel cells, it did not contribute to SK activation.

The PMCA pumps in genetically determined neuronal pathologies.

Ca2+ signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca2+ must thus be very precisely regulated: in all cells Ca2+ pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca2+ ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca2+ ATPase and the Secretory Pathway Ca2+ ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na+/Ca2+ exchanger (NCX) also exports Ca2+ from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca2+. However, they are important in the regulation of Ca2+ in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Ca2+the C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca2+ increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca2+ signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their decreased ability to export Ca2+. The defect in the bulk cytosolic Ca2+ homeostasis is minor, in keeping with the role of the PMCA pumps in the local control of Ca2+ in specialized plasma membrane microdomains.

The Plasma Membrane Calcium Pump (PMCA): Regulation of Cytosolic Ca2+, Genetic Diversities and Its Role in Sub-plasma Membrane Microdomains.

In this chapter the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to the structural and functional properties of PMCA, the tissue distribution of the different isoforms, including their differences during development. The importance of PMCA for regulating Ca2+ signaling in microdomains under different conditions is also discussed.

IP3 Receptor Properties and Function at Membrane Contact Sites.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a ubiquitously expressed Ca2+-release channel localized in the endoplasmic reticulum (ER). The intracellular Ca2+ signals originating from the activation of the IP3R regulate multiple cellular processes including the control of cell death versus cell survival via their action on apoptosis and autophagy. The exact role of the IP3Rs in these two processes does not only depend on their activity, which is modulated by the cytosolic composition (Ca2+, ATP, redox status, …) and by various types of regulatory proteins, including kinases and phosphatases as well as by a number of oncogenes and tumor suppressors, but also on their intracellular localization, especially at the ER-mitochondrial and ER-lysosomal interfaces. At these interfaces, Ca2+ microdomains are formed, in which the Ca2+ concentration is finely regulated by the different ER, mitochondrial and lysosomal Ca2+-transport systems and also depends on the functional and structural interactions existing between them. In this review, we therefore discuss the most recent insights in the role of Ca2+ signaling in general, and of the IP3R in particular, in the control of basal mitochondrial bioenergetics, apoptosis, and autophagy at the level of inter-organellar contact sites.

ITPRs/inositol 1,4,5-trisphosphate receptors in autophagy: From enemy to ally.

ITPRs (inositol 1,4,5-trisphosphate receptors), the main endoplasmic reticulum (ER) Ca(2+)-release channels, were originally proposed as suppressors of autophagy. Yet, new evidence has accumulated over recent years supporting a crucial, stimulatory role for ITPRs in driving the autophagic flux. Here, we provide an integrated view on how ITPR-mediated Ca(2+) signaling can have a dual impact on autophagy, depending on the characteristics of the spatio-temporal Ca(2+) signals, including the existence of ER-mitochondrial and ER-lysosomal Ca(2+) signaling microdomains.