Comparative analysis of optogenetic actuators in cultured astrocytes

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d-serine and l-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca²⁺ ([Ca²⁺]_i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca²⁺ permeability (Ca²⁺ translocating channelrhodopsin, CatCh). We show here that [Ca²⁺]_i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca²⁺ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca²⁺]_i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G_s-coupled) resulted in astrocytic [Ca²⁺]_i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca²⁺]_i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.     

The low conductance mitochondrial permeability transition pore confers excitability and CICR wave propagation in a computational model

Mitochondria have long been known to sequester cytosolic Ca²⁺ and even to shape intracellular patterns of endoplasmic reticulum-based Ca²⁺ signaling. Evidence suggests that the mitochondrial network is an excitable medium which can demonstrate independent Ca²⁺ induced Ca²⁺ release via the mitochondrial permeability transition. The role of this excitability remains unclear, but mitochondrial Ca²⁺ handling appears to be a crucial element in diverse diseases as diabetes, neurodegeneration and cardiac dysfunction that also have bioenergetic components. In this paper, we extend the modular Magnus-Keizer computational model for respiration-driven Ca²⁺ handling to include a permeability transition based on a channel-like pore mechanism. We demonstrate both excitability and Ca²⁺ wave propagation accompanied by depolarizations qualitatively similar to those reported in cell and isolated mitochondria preparations. These waves depend on the energy state of the mitochondria, as well as other elements of mitochondrial physiology. Our results support the concept that mitochondria can transmit state dependent signals about their function across the mitochondrial network. Our model provides the tools for predictions about the internal physiology that leads to this qualitatively different Ca²⁺ excitability seen in mitochondria.     

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca²⁺) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca²⁺ signaling is essential for the cellular and developmental changes that support T. gondii parasitism.The characterization of the molecular players and pathways directly activated by Ca²⁺ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca²⁺ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca²⁺ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.     

Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca response in teleost embryo

Intracellular Ca²⁺ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca²⁺ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca²⁺ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca²⁺ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca²⁺ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca²⁺ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca²⁺ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca²⁺ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca²⁺ imaging in vivo.     

Bcl-2 inhibitors as anti-cancer therapeutics: The impact of and on calcium signaling

Bcl-2-protein family members are essential regulators of apoptosis. Anti-apoptotic Bcl-2 proteins ensure cell survival via different mechanisms, including via binding of pro-apoptotic Bcl-2-family members and the modulation of intracellular Ca²⁺-transport systems. Many cancer cells upregulate these proteins to overcome the consequences of ongoing oncogenic stress. Bcl-2 inhibition leading to cell death, therefore emerged as a novel cancer therapy. Different Bcl-2 inhibitors have already been developed including the hydrophobic cleft-targeting BH3 mimetics, which antagonize Bcl-2’s ability to scaffold and neutralize pro-apoptotic Bcl-2-family members. As such, the BH3 mimetics have progressed into clinical studies as precision medicines. Furthermore, new inhibitors that target Bcl-2’s BH4 domain have been developed as promising anti-cancer tools. Given Bcl-2’s role in Ca²⁺ signaling, these drugs and tools can impact Ca²⁺ signaling. In addition to this, some Bcl-2 inhibitors may have “off-target” effects that cause Ca²⁺-signaling dysregulation not only in cancer cells but also in healthy cells, resulting in adverse effects. In this review, we aim to provide an up-to-date overview of the involvement of intracellular Ca²⁺ signaling in the working mechanism and “off-target” effects of the different Bcl-2-antagonizing small molecules and peptides.     

Direct control of store-operated calcium channels by ultrafast laser

Ca²⁺ channels are essential to cell birth, life, and death. They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca²⁺ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca²⁺ influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca²⁺ influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.Subject terms: Biological techniques, Ion channel signalling, Calcium signalling     

Calcium signaling in lacrimal glands

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca²⁺-mobilizing messenger, IP₃, and release of Ca²⁺ stored in the endoplasmic reticulum. The loss of Ca²⁺ from the endoplasmic reticulum then triggers a process known as store-operated Ca²⁺ entry, involving a Ca²⁺ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Using aequorin probes to measure Ca2+ in intracellular organelles

Aequorins are excellent tools for measuring intra-organellar Ca²⁺ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca²⁺ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca²⁺ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca²⁺ indicator for monitoring physiological Ca²⁺ signaling ex vivo and in vivo.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

Spontaneous calcium transients in the immature adult-born neurons of the olfactory bulb

Spontaneous neuronal activity and concomitant intracellular Ca²⁺ signaling are abundant during early perinatal development and are well known for their key role in neuronal proliferation, migration, differentiation and wiring. However, much less is known about the in vivo patterns of spontaneous Ca²⁺ signaling in immature adult-born cells. Here, by using two-photon Ca²⁺ imaging, we analyzed spontaneous in vivo Ca²⁺ signaling in adult-born juxtaglomerular cells of the mouse olfactory bulb over the time period of 5 weeks, from the day of their arrival in the glomerular layer till their stable integration into the preexisting neural network. We show that spontaneous Ca²⁺ transients are ubiquitously present in adult-born cells right after their arrival, require activation of voltage-gated Na⁺ channels and are little sensitive to isoflurane anesthesia. Interestingly, several parameters of this spontaneous activity, such as the area under the curve, the time spent in the active state as well as the fraction of continuously active cells show a bell-shaped dependence on cell’s age, all peaking in 3–4 weeks old cells. This data firmly document the in vivo presence of spontaneous Ca²⁺ signaling during the layer-specific maturation of adult-born neurons in the olfactory bulb and motivate further analyses of the functional role(s) of this activity.     

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca²⁺ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.     

Modeling local and global intracellular calcium responses mediated by diffusely distributed inositol 1,4,5-trisphosphate receptors

Considerable insight into intracellular Ca²⁺ responses has been obtained through the development of whole cell models that are based on molecular mechanisms, e.g., single channel kinetics of the inositol 1,4,5-trisphosphate (IP₃) receptor Ca²⁺ channel. However, a limitation of most whole cell models to date is the assumption that IP₃ receptor Ca²⁺ channels (IP₃Rs) are globally coupled by a “continuously stirred” bulk cytosolic [Ca²⁺], when in fact open IP₃Rs experience elevated “domain” Ca²⁺ concentrations. Here we present a 2N+2-compartment whole cell model of local and global Ca²⁺ responses mediated by N=100,000 diffusely distributed IP₃Rs, each represented by a four-state Markov chain. Two of these compartments correspond to bulk cytosolic and luminal Ca²⁺ concentrations, and the remaining 2N compartments represent time-dependent cytosolic and luminal Ca²⁺ domains associated with each IP₃R. Using this Monte Carlo model as a starting point, we present an alternative formulation that solves a system of advection-reaction equations for the probability density of cytosolic and luminal domain [Ca²⁺] jointly distributed with IP₃R state. When these equations are coupled to ordinary differential equations for the bulk cytosolic and luminal [Ca²⁺], a realistic but minimal model of whole cell Ca²⁺ dynamics is produced that accounts for the influence of local Ca²⁺ signaling on channel gating and global Ca²⁺ responses. The probability density approach is benchmarked and validated by comparison to Monte Carlo simulations, and the two methods are shown to agree when the number of Ca²⁺ channels is large (i.e., physiologically realistic). Using the probability density approach, we show that the time scale of Ca²⁺ domain formation and collapse (both cytosolic and luminal) may influence global Ca²⁺ oscillations, and we derive two reduced models of global Ca²⁺ dynamics that account for the influence of local Ca²⁺ signaling on global Ca²⁺ dynamics when there is a separation of time scales between the stochastic gating of IP₃Rs and the dynamics of domain Ca²⁺.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.     

Neuronal calcium signaling via store-operated channels in health and disease

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca²⁺) into cells in response to the depletion of intracellular Ca²⁺ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca²⁺ sensor STIM1 and plasma membrane Ca²⁺ channel Orai1 and serves to sustain Ca²⁺ signaling and refill ER Ca²⁺ stores. In contrast, because of the complexity of Ca²⁺ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target.     

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

Calcium signaling in osteoclasts

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca²⁺) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca²⁺ oscillations occur during RANKL-mediated osteoclastogenesis. Ca²⁺ oscillations provide a digital Ca²⁺ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca²⁺/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca²⁺ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca²⁺ oscillations, and we discuss possible molecular players in this specialized Ca²⁺ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Mechanisms for L-channel-mediated increase in [Ca]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels

The importance of Ca²⁺ signaling in astrocytes is undisputed but a potential role of Ca²⁺ influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in astrocytes in primary cultures in response to an increased extracellular K⁺ concentration (45 mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca²⁺, known to occur in response to an elevation in [Ca²⁺]_i. This means that the actual influx of Ca²⁺ is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca²⁺ currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K⁺-mediated increase in [Ca²⁺]_i. This is shown to be due to an inhibition of capacitative Ca²⁺ influx, reflected by decreased mRNA and protein expression of the ‘transient receptor potential channel’ (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca²⁺ influx in response to an elevated extracellular K⁺ concentration is important for generation of Ca²⁺ oscillations and for the stimulatory effect of elevated K⁺ concentrations in intact, non-cultured brain tissue, and (ii) that Ca²⁺ channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Ca_v1.3 is demonstrated in freshly separated astrocytes from normal brain.     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.