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Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes
Journal of Neurochemistry
Abstract
Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.
... The TRPC subfamily has seven members in mammals (TRPC1-7) which participate in storeoperated calcium entry (SOCE) and/or receptor-operated calcium entry (ROCE) in various cell types, including astrocytes, and play a critical role in many cellular processes [14]. TRPC1-TRPC6 are expressed in cultured embryonic astrocytes [15,16]. TRPC1 and TRPC3 likely contribute to SOCE in astrocytes, induced by endoplasmic reticulum depletion or metabotropic stimulation [17,18]. ...
... Subtle and temporal regulation of astrocyte functions after stroke will undoubtedly impact the survival of neurons. TRPC1-TRPC6 are expressed in cultured embryonic astrocytes [15,16]. The present study was designed to elucidate the role of the TRPC3/6/7 group of TRPC channels in brain ischemic injury, focusing on astrocytes in cerebral ischemia reperfusion injury. ...
... The TRPC family proteins can be responsible for SOCE and ROCE in a variety of cell types [34]. According to recent findings, Ca 2+ entry through receptor-operated Ca 2+ channels (ROCs) and store-operated Ca 2+ channels (SOCs) contributes to shape cytoplasmic Ca 2+ signals in astrocytes [15,17,35]. ...
Ischemia contributes significantly to morbidity and mortality associated with many common neurological diseases. Calcium overload is an important mechanism of cerebral ischemia and reperfusion (I/R) injury. Despite decades of intense research, an effective beneficial treatment of stroke remains limited; few therapeutic strategies exist to combat the consequences of cerebral ischemia. Traditionally, a "neurocentric" view has dominated research in this field. Evidence is now accumulating that glial cells, especially astrocytes, play an important role in the pathophysiology of cerebral ischemia. Here, we show that transient receptor potential (TRP)C3/6/7 knockout (KO) mice subjected to an I/R procedure demonstrate ameliorated brain injury (infract size), compared to wild-type (WT) control animals. This is accompanied by reduction of NF-кB phosphorylation and an increase in protein kinase B (AKT) phosphorylation in I/R-injured brain tissues in TRPC3/6/7 KO mice. Also, the expression of pro-apoptotic protein Bcl-2 associated X (Bax) is down-regulated and that of anti-apoptotic protein Bcl-2 is upregulated in TRPC3/6/7(-/-) mice. Astrocytes isolated from TRPC3/6/7 KO mice and subjected to oxygen/glucose deprivation and subsequent reoxygenation (OGD-R, mimicking in vivo I/R injury) also exhibit enhanced Bcl-2 expression, reduced Bax expression, enhanced AKT phosphorylation, and reduced NF-кB phosphorylation. Furthermore, apoptotic rates of TRPC3/6/7 KO astrocytes cultured in OGD-R conditions were reduced significantly compared to WT control. These findings suggest TRPC3/6/7 channels play a detrimental role in brain I/R injury. Deletion of these channels can interfere with the activation of NF-кB (pro-apoptotic), promote activation of AKT (anti-apoptotic), and ultimately, ameliorate brain damage via inhibition of astrocyte apoptosis after cerebral ischemia/reperfusion injury.
... Noteworthy, in culture of rat cortical astrocytes at higher concentration of agonist additional Ca 2+ influx pathway activated by glutamate was found. This influx pathway, attributable to AMPA/kainite receptors, is sensitive to NBQX but insensitive to SOCE blocker La 3+ (Pizzo et al., 2001). In the present study, we found that SOCE in neurons was approximately two-times lower than in glial cells. ...
... This likely reflects the amount and type of AMPARs in both types of cells (Figure 3B; Verkhratsky and Steinh?user, 2000). Although glial cells possess functional AMPARs (Pizzo et al., 2001; Lalo et al., 2006), the glial population does not contribute significantly to the total amount of AMPARs when analyzed by Western blot and real-time polymerase chain reaction (Orlandi et al., 2011). The expression of all four AMPA subunits was detected in rat cortical glia, but the GluA2 subunit was most abundant (Holzwarth et al., 1994). ...
... However, astrocytes have lower expression of Ca 2+ -permeable AMPARs with Q/R-unedited GluR2 subunits than neurons (Whitney et al., 2008). In cortical astrocytes, AMPARs are stimulated by glutamate and AMPA and can be inhibited by NBQX (Pizzo et al., 2001; Lalo et al., 2011). Glutamate-induced Ca 2+ influx in rat cortical astrocytes was also shown to be sensitive to LaCl 3 , which inhibits SOCE (Pizzo et al., 2001). ...
The process of store-operated calcium entry (SOCE) leads to refilling the endoplasmic reticulum (ER) with calcium ions (Ca²⁺) after their release into the cytoplasm. Interactions between (ER)-located Ca²⁺ sensors (stromal interaction molecule 1 [STIM1] and STIM2) and plasma membrane-located Ca²⁺ channel-forming protein (Orai1) underlie SOCE and are well described in non-excitable cells. In neurons, however, SOCE appears to be more complex because of the importance of Ca²⁺ influx via voltage-gated or ionotropic receptor-operated Ca²⁺ channels. We found that the SOCE inhibitors ML-9 and SKF96365 reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca²⁺]i amplitude by 80% and 53%, respectively. To assess the possible involvement of AMPA receptors (AMPARs) in SOCE, we used their specific inhibitors. As estimated by Fura-2 acetoxymethyl (AM) single-cell Ca²⁺ measurements in the presence of CNQX or NBQX, thapsigargin (TG)-induced Ca²⁺ influx decreased 2.2 or 3.7 times, respectively. These results suggest that under experimental conditions of SOCE when Ca²⁺ stores are depleted, Ca²⁺ can enter neurons also through AMPARs. Using specific antibodies against STIM proteins or GluA1/GluA2 AMPAR subunits, co-immunoprecipitation assays indicated that when Ca²⁺ levels are low in the neuronal ER, a physical association occurs between endogenous STIM proteins and endogenous AMPAR receptors. Altogether, our data suggest that STIM proteins in neurons can control AMPA-induced Ca²⁺ entry as a part of the mechanism of SOCE.
... The astroglial cell line U373 MG has been found to express both Orai and STIM1 proteins, although their direct association with store-operated Ca 2þ influx needs further scrutiny (Barajas et al., 2008). The CRAC channels, which produce Ca 2þ -release activated Ca 2þ currents (I CRAC ) in several types of nonexcitable cell, were not detected in astrocytes; there are, however, certain similarities in the pharmacological profiles of SOCE in astroglia and in the rat basophilic leukemia cell line (Pizzo et al., 2001). ...
... TRP channels are expressed in astroglia (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001), and there are experimental indications that TRP channels participate in Ca 2þ signal generation in astrocytes in vitro (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001). Astrocytes, both freshly isolated and in culture, were found to express canonical type TRP (TRPC) channels assembled from an obligatory TRPC1 combined with ancillary TRPC4 and/or TRPC5 proteins (Golovina, 2005;Malarkey et al., 2008). ...
... TRP channels are expressed in astroglia (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001), and there are experimental indications that TRP channels participate in Ca 2þ signal generation in astrocytes in vitro (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001). Astrocytes, both freshly isolated and in culture, were found to express canonical type TRP (TRPC) channels assembled from an obligatory TRPC1 combined with ancillary TRPC4 and/or TRPC5 proteins (Golovina, 2005;Malarkey et al., 2008). ...
Astrocytes are highly diverse and heterogeneous neuroglial cells which represent the main homeostatic element of the central nervous system. They are capable of sensing (through a wide variety of receptors) and secreting neurotransmitters and neuromodulators and are therefore incorporated into the chemical transmission system operating in neural networks. Astrocytes possess a specific form of excitability provided by movements of calcium ions (Ca 2+ ) between intracellular compartments and plasmalemmal Ca 2+ fluxes. This “Ca 2+ excitability” is controlled by several families of evolutionarily conserved proteins localized in the plasmalemma, within the cytosol and in the endomembranes. Astroglial Ca 2+ signals can cross intercellular boundaries, creating propagating Ca 2+ waves that enable long-range signaling in astroglial networks.
... The astroglial cell line U373 MG has been found to express both Orai and STIM1 proteins, although their direct association with store-operated Ca 2þ influx needs further scrutiny (Barajas et al., 2008). The CRAC channels, which produce Ca 2þ -release activated Ca 2þ currents (I CRAC ) in several types of nonexcitable cell, were not detected in astrocytes; there are, however, certain similarities in the pharmacological profiles of SOCE in astroglia and in the rat basophilic leukemia cell line (Pizzo et al., 2001). ...
... TRP channels are expressed in astroglia (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001), and there are experimental indications that TRP channels participate in Ca 2þ signal generation in astrocytes in vitro (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001). Astrocytes, both freshly isolated and in culture, were found to express canonical type TRP (TRPC) channels assembled from an obligatory TRPC1 combined with ancillary TRPC4 and/or TRPC5 proteins (Golovina, 2005;Malarkey et al., 2008). ...
... TRP channels are expressed in astroglia (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001), and there are experimental indications that TRP channels participate in Ca 2þ signal generation in astrocytes in vitro (Golovina, 2005;Grimaldi et al., 2003;Pizzo et al., 2001). Astrocytes, both freshly isolated and in culture, were found to express canonical type TRP (TRPC) channels assembled from an obligatory TRPC1 combined with ancillary TRPC4 and/or TRPC5 proteins (Golovina, 2005;Malarkey et al., 2008). ...
Astrocytes are the most diverse and highly heterogeneous neuroglial cells which represent the main homeostatic element of the central nervous system. Astrocytes are capable of sensing (through a wide variety of receptors) and secreting neurotransmitters and neuromodulators being thus incorporated in the chemical transmission system operating in neural networks. Astrocytes possess a specific form of excitability provided by movements of Ca2+ ions between intracellular compartments and plasmalemmal Ca2+ fluxes. This “Ca2+ excitability” is controlled by several families of evolutionary conserved proteins localised in the plasmalemma, within the cytosol and in the endomembranes. Astroglial Ca2+ signals can cross intercellular boundaries creating propagating Ca2+ waves that serve for long-range signalling in astroglial networks.
... Since in astrocytes MLC1 is activated by stimuli (hyposmotic, inflammatory) inducing intracellular calcium release from the ER compartment, and the consequent capacitative calcium entry (CCE) needed for ER refilling (Fischer et al., 1997), we optimized an experimental protocol to distinguish between ER calcium release and CCE as potential intracellular calcium source for MLC1 activation. We stimulated cells with thapsigargin (TG), the non-competitive inhibitor of the ER Ca 2+ ATPase pumps (SERCA), in the presence or not of the CCE inhibitor lanthanum chloride (LaCl 3 ), (Pizzo et al., 2001). SERCA pump drives cytosolic free Ca 2+ ion entrance into the ER lumen, and its inhibition causes a massive Ca 2+ release from the ER followed by CCE. ...
... A similar increase was observed when U251 cells were treated with the P2Y purinergic receptors agonist ATP (100 μM for 5 min, Fig. 1B), capable of triggering InsP3-mediated Ca 2+ release from the ER (Pizzo et al., 2001). Accordingly, IF images of ATP-treated cells showed higher levels of MLC1 at PM when compared to untreated cells (Fig. 1C, E, F), confirming the main role of ER Ca 2+ release on MLC1 protein assembly and sorting. ...
... In neurons, ORAI1 appears dispensable for SOCE [46], but it has been implicated in neuronal excitability [45,48], whereas ORAI2 seems to be part of a neuronal SOC (nSOC) based on TRPC6 and activated by STIM2, and its activity is impaired in mouse AD models [46]. In glial cells, SOCE plays a major role in the Ca 2+ -based excitability that characterizes these cells both in culture [14,42,47,49] and in situ [50,51] studies. ...
... In neurons, the identification of both SOCE components and role is still controversial [43], while SOCE seems to play a primary role in modulating the Ca 2+ -based excitability in astrocytes [47,49,51,71]. It is worth noting that astrocytes are also directly involved in Aβ production [26,[55][56][57][58], while alterations in Ca 2+ -based excitability has been reported in astrocytes from FAD mouse models [54,72,73]. ...
Senile plaques, the hallmarks of Alzheimer's Disease (AD), are generated by the deposition of amyloid-beta (Aβ), the proteolytic product of amyloid precursor protein (APP), by β and γ-secretase. A large body of evidence points towards a role for Ca2+ imbalances in the pathophysiology of both sporadic and familial forms of AD (FAD). A reduction in store-operated Ca2+ entry (SOCE) is shared by numerous FAD-linked mutations, and SOCE is involved in Aβ accumulation in different model cells. In neurons, both the role and components of SOCE remain quite obscure, whereas in astrocytes, SOCE controls their Ca2+-based excitability and communication to neurons. Glial cells are also directly involved in Aβ production and clearance. Here, we focus on the role of ORAI2, a key SOCE component, in modulating SOCE in the human neuroglioma cell line H4. We show that ORAI2 overexpression reduces both SOCE level and stores Ca2+ content, while ORAI2 downregulation significantly increases SOCE amplitude without affecting store Ca2+ handling. In Aβ-secreting H4-APPswe cells, SOCE inhibition by BTP2 and SOCE augmentation by ORAI2 downregulation respectively increases and decreases Aβ42 accumulation. Based on these findings, we suggest ORAI2 downregulation as a potential tool to rescue defective SOCE in AD, while preventing plaque formation.
... SOCE is a common feature present in all types of astrocytes (Tuschick et al., 1997;Pivneva et al., 2008;Verkhratsky & Parpura, 2014). Although all the components of the SOCE molecular machinery, including STIM, Orai and TRP channels (Pizzo et al., 2001;Grimaldi et al., 2003;Golovina, 2005;Barajas et al., 2008;Malarkey et al., 2008;Ronco et al., 2014;, are present in astrocytes, experiments suggest that astroglial cells employ mainly TRP channels to generated SOCE (Verkhratsky & Parpura, 2014) and to coordinate ion signalling and astroglial excitability . Intriguingly, SOCE is the main mechanism for CaN activation in peripheral immune cells and lymphocytes (Gwack et al., 2007;Oh-hora & Rao, 2008). ...
... The mechanisms of CICR and SOCE have been generally implicated in setting up the oscillatory Ca 2+ dynamics (Carafoli et al., 2001;Uhlen & Fritz, 2010;Dupont et al., 2011;Parekh, 2011). Specifically in astrocytes, TRP channels are involved in glutamate-induced Ca 2+ oscillations (Pizzo et al., 2001). Ca 2+ -dependent release of ATP, instead, is implicated in synchronized Ca 2+ oscillations in astroglial networks (Koizumi, 2010). ...
Astrocytes perform fundamental housekeeping functions in the central nervous system and through bidirectional communication with neurons are thought to coordinate synaptic transmission and plasticity. They are also renowned actors in brain pathology. Reactive gliosis and neuroinflammation are featured by many (if not all) acute and chronic neurodegenerative pathologies including Alzheimer’s disease (AD). The Ca2+/calmodulin-activated phosphatase calcineurin (CaN) plays a central role in the pathology-related changes of astroglial cells mainly through activation of the inflammation-related transcription factors Nuclear Factor of Activated T-cells (NFAT) and Nuclear Factor kB (NF-kB). In this contribution we focus on the mechanistic aspects of CaN signalling in astrocytes. We analyze the astroglial Ca2+ signalling toolkit in the context of Ca2+ signals necessary for CaN activation and focus on the astroglial CaN signalling through its direct target, NFAT, as well as the intricate relationships between CaN and NF-kB activation pathways.The majority of data about CaN-mediated signalling in astrocytes point to the role for CaN in pathology-related conditions while very little is currently known about signalling and function of astroglial CaN in physiology.
... Embryonic cultured astrocytes (also often referred to as astrocytes type I) express mRNA for TRPC1 to TRPC6 (Grimaldi et al. 2003;Pizzo et al. 2001) and were reported to produce Ca 2+ fluxes and [Ca 2+ ] i oscillations in response to oleyl-acetylglycerol (an analogue of DAG) and following stimulation of glutamate receptors and endoplasmic reticulum (ER) store depletion. In spinal astrocytes, the mRNAs for TRPC1, 2, 3, 4 and 6 were detected (Miyano et al. 2010). ...
... Channels formed by Orai and TRPC have distinct biophysical identity and their corresponding currents and functional responses can be easily distinguished. This SOCE pathway is functioning in virtually all types of neuroglial cells (Hartmann and Verkhratsky 1998;Malarkey et al. 2008;Moller et al. 1997;Muller et al. 2013;Paez et al. 2009;Pivneva et al. 2008;Pizzo et al. 2001;Reyes and Parpura 2009;Toescu et al. 1998;Tuschick et al. 1997). To the best of our knowledge, characteristic I CRAC channels have not been hitherto recorded from mature astrocytes and evidence about functional operation of Orai/STIM complex derives from neoplastic cell lines and astrocytes in vitro (Moreno et al. 2012;Motiani et al. 2013). ...
Astroglial excitability is based on highly spatio-temporally coordinated fluctuations of intracellular ion concentrations, among which changes in Ca(2+) and Na(+) take the leading role. Intracellular signals mediated by Ca(2+) and Na(+) target numerous molecular cascades that control gene expression, energy production and numerous homeostatic functions of astrocytes. Initiation of Ca(2+) and Na(+) signals relies upon plasmalemmal and intracellular channels that allow fluxes of respective ions down their concentration gradients. Astrocytes express several types of TRP channels of which TRPA1 channels are linked to regulation of functional expression of GABA transporters, whereas TRPV4 channels are activated following osmotic challenges and are up-regulated in ischaemic conditions. Astrocytes also ubiquitously express several isoforms of TRPC channels of which heteromers assembled from TRPC1, 4 and/or 5 subunits that likely act as stretch-activated channels and are linked to store-operated Ca(2+) entry. The TRPC channels mediate large Na(+) fluxes that are associated with the endoplasmic reticulum Ca(2+) signalling machinery and hence coordinate Na(+) and Ca(2+) signalling in astroglia.
... However, in astrocytes the studies about the molecular machinery underlying SOCE have focused mainly in the role of TRPCs. There is evidence supporting the participation of TRPC1 in SOCE in mouse cortical astrocytes treated with CPA [39], and in rat cortical astrocytes stimulated with glutamate [40]. Moreover, TRPC3 underlies SOCE in rat spinal astrocytes when activated by substance P [41]. ...
... In addition, the residual STIM1 that remains after the knockdown could be sufficient to activate other SOCs, such as TRPCs. Rat astrocytes express all the members of the TRPC family [40] . In astrocytes, the TRPC1 is responsible of 40% of the CPAinduced SOCE and almost all the Ca 2+ influx evoked by purinergic stimulation [65]. ...
In astrocytes, thrombin leads to cytoplasmic Ca(2+) elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca(2+) increase generated by an interplay between ER-Ca(2+) release and store-operated Ca(2+) entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca(2+) depletion and binds Orai1 to activate Ca(2+) influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca(2+) response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca(2+) influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca(2+) influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca(2+) response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca(2+) release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca(2+) influx.
... An alternative strategy is to act on SOCE, a mechanism of Ca 2+ influx across the PM to the ER operating in different cell types, including astrocytes 72 . It is well known that SOCE is necessary for ER-mediated Ca 2+ signaling in astrocytes [73][74][75] and it is also implicated in gliotransmitter release in the hippocampus 63 . SOCE machinery is localized at ER-PM junctions and involves the concerted action of STIM1, the Ca 2+ sensor monitoring intraluminal [Ca 2+ ] ER , and Orai and/or TRPC channels on the PM. ...
Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer’s disease (AD) linking astrocyte Ca²⁺ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca²⁺ signaling, closely associated with decreased endoplasmic reticulum Ca²⁺ concentration and reduced expression of the Ca²⁺ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca²⁺ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca²⁺ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.
... Moreover, to assess if also CCE has a role on MLC1 stimulation, the above treatments were performed in absence or presence of lanthanum chloride (LaCl3), inhibits CCE by acting on PM Ca 2+permeant channels [43]. WB of protein extracted from U251 cells after a 5 min stimulation with 100 nM of TG (experimental conditions causing a rapid and sustained Ca 2+ release in glioma cells) [44], showed an increase of the MLC1 dimeric, membrane associated form when compared to unstimulated cells (Fig. 1C, arrow). ...
MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.
... Spontaneous astrocyte MCEs that occur in the absence of synaptic activity have been shown to be mediated by mitochondrial Ca 2+ release [14] via the opening of mitochondrial permeability transition pore [15] and by extracellular Ca 2+ influx through transient receptor potential cation channel A1 (TRPA1) [20,25]. It should be noted that other TRP channels such as TRPV1, TRPV4, TRPC1, TRPC3, TRPC4, and TRPC5 may also mediate Ca 2+ influx in astrocytes [83][84][85][86][87][88], but there is limited evidence that these channels are directly activated during synaptic transmission. ...
Astrocytes are complex glial cells that play many essential roles in the brain, including the fine-tuning of synaptic activity and blood flow. These roles are linked to fluctuations in intracellular Ca2+ within astrocytes. Recent advances in imaging techniques have identified localized Ca2+ transients within the fine processes of the astrocytic structure, which we term microdomain Ca2+ events. These Ca2+ transients are very diverse and occur under different conditions, including in the presence or absence of surrounding circuit activity. This complexity suggests that different signalling mechanisms mediate microdomain events which may then encode specific astrocyte functions from the modulation of synapses up to brain circuits and behaviour. Several recent studies have shown that a subset of astrocyte microdomain Ca2+ events occur rapidly following local neuronal circuit activity. In this review, we consider the physiological relevance of microdomain astrocyte Ca2+ signalling within brain circuits and outline possible pathways of extracellular Ca2+ influx through ionotropic receptors and other Ca2+ ion channels, which may contribute to astrocyte microdomain events with potentially fast dynamics.
... Canonical SOCE requires two molecular components, stromal interacting molecule (STIM) and Orai channels, although, transient receptor potential (TRP) channels are also subject of STIM regulation (Cheng et al., 2013;Salido et al., 2009). All components of the SOCE molecular machinery, including STIM1,2, Orai1,2,3 and TRP channels have been detected in astrocytes (Golovina, 2005;Grimaldi et al., 2003;Malarkey et al., 2008;Pizzo et al., 2001;Ronco et al., 2014). Experiments with anti-TRPC1 antibodies suggest pivotal role of TRP channels (Malarkey et al., 2008;Reyes et al., 2013;Verkhratsky and Nedergaard, 2018), however evidence indicating substantial role for ORAI channels in astroglial SOCE are on the rise (Kwon et al., 2017;Toth et al., 2019). ...
Glial cells exploit calcium (Ca²⁺) signals to perceive the information about the activity of the nervous tissue and the tissue environment to translate this information into an array of homeostatic, signaling and defensive reactions. Astrocytes, the best studied glial cells, use several Ca²⁺ signaling generation pathways that include Ca²⁺ entry through plasma membrane, release from endoplasmic reticulum (ER) and from mitochondria. Activation of metabotropic receptors on the plasma membrane of glial cells is coupled to an enzymatic cascade in which a second messenger, InsP3 is generated thus activating intracellular Ca²⁺ release channels in the ER endomembrane. Astrocytes also possess store-operated Ca²⁺ entry and express several ligand-gated Ca²⁺ channels. In vivo astrocytes generate heterogeneous Ca²⁺ signals, which are short and frequent in distal processes, but large and relatively rare in soma. In response to neuronal activity intracellular and inter-cellular astrocytic Ca²⁺ waves can be produced. Astrocytic Ca²⁺ signals are involved in secretion, they regulate ion transport across cell membranes, and are contributing to cell morphological plasticity. Therefore, astrocytic Ca²⁺ signals are linked to fundamental functions of the central nervous system ranging from synaptic transmission to behavior. In oligodendrocytes, Ca²⁺ signals are generated by plasmalemmal Ca²⁺ influx, or by release from intracellular stores, or by combination of both. Microglial cells exploit Ca²⁺ permeable ionotropic purinergic receptors and transient receptor potential channels as well as ER Ca²⁺ release. In this contribution, basic morphology of glial cells, glial Ca²⁺ signaling toolkit, intracellular Ca²⁺ signals and Ca²⁺-regulated functions are discussed with focus on astrocytes.
... Although the role of SOCE in neurons is still unclear, it is important to stress that, in excitable cells, STIM and ORAI components might also play non-canonical roles-STIM1 binds to L-type VOCCs, inhibits their gating, and induces channel internalization [129,130] while ORAI1 increases neuronal excitability [131,132]. At variance with neuronal cells, it is now largely accepted that SOCE is crucial for the Ca 2+ -based excitability that characterizes glial cells both in vitro [133,134] and in vivo [135]. Nonetheless, studies that specifically address the role of FAD-PS2 in glial SOCE modulation are still lacking. ...
Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-β (Aβ) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca 2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca 2+ handling to Ca 2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca 2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).
... In astrocytes, which are the most abundant cells in the brain, several isoforms of TRP are expressed. TRPC1-6 have been detected in cultured astrocytes with quantitative differences [31]. In fact, knockdown of the TRPC6 gene markedly reduced receptor-operated Ca 2+ entry [32]. ...
Transient receptor potential (TRP) proteins consist of a superfamily of cation channels that have been involved in diverse physiological processes in the brain as well as in the pathogenesis of neurological disease. TRP channels are widely expressed in the brain, including neurons and glial cells, as well as in the cerebral vascular endothelium and smooth muscle. Members of this channel superfamily show a wide variety of mechanisms ranging from ligand binding to voltage, physical, and chemical stimuli, implying the promising therapeutic potential of TRP in neurological diseases. In this review, we focus on the physiological functions of TRP channels in the brain and the pathological roles in neurological disorders to explore future potential neuroprotective strategies.
... Listed stimuli could evoke Ca 2+ signalling by activating transient receptor potential channels (reviewed in Verkhratsky and Burnstock, 2014). TRP channels expressed in astrocytes (TRPA-1, TRPV-4, TRPC-1, 4, 5) (Pizzo et al., 2001;Golovina, 2005;Song et al., 2005) regulate the Ca 2+ basal level and contribute to spontaneous Ca 2+ transients Shigetomi and Jackson-Weaver, et al., 2013), thus affecting the store-operated Ca 2+ entry (Golovina, 2005). ...
It is widely recognised that astrocytes are able to shape synaptic transmission by restricting glutamate transients to the synaptic cleft. In this thesis, I demonstrate that during synaptic transmission K+ efflux through postsynaptic NMDA receptors depolarises the astrocytic membrane and thus slows down glial glutamate uptake. This effect involves the rectifying K+ channels (Kir4.1), predominantly located at perisynaptic astrocytic processes (PAPs). Genetic upregulation of this channel subtype in astrocytes does not affect glutamate transporters efficiency but curtails increase in presynaptic glutamate release probability during extracellular K+ rises. Thus, activity-dependent accumulation of extracellular K+ can boost glutamate release from the presynaptic site while decreasing astroglial glutamate uptake. Both factors occasion increased extrasynaptic glutamate escape and therefore inter-synaptic crosstalk in the hippocampus.
... Functional expression of TRPA1 was also demonstrated in a subpopulation of hippocampal astrocytes [360,362]. The TRPC ('canonical') channels were detected in freshly isolated and in primary cultured astrocytes, which were reported to express all subtypes of these channels from TRPC1 to TRPC6 [124, 244,308]. These TRPC channels contribute to astroglial Ca 2+ signalling induced by purinergic, glutamatergic and mechanical stimulation [231,323,324]. ...
Astrocytes are principal cells responsible for maintaining the brain homeostasis. Additionally, these glial cells are also involved in homocellular (astrocyte-astrocyte) and heterocellular (astrocyte-other cell types) signalling and metabolism. These astroglial functions require an expression of the assortment of molecules, be that transporters or pumps, to maintain ion concentration gradients across the plasmalemma and the membrane of the endoplasmic reticulum. Astrocytes sense and balance their neurochemical environment via variety of transmitter receptors and transporters. As they are electrically non-excitable, astrocytes display intracellular calcium and sodium fluctuations, which are not only used for operative signalling but can also affect metabolism. In this chapter we discuss the molecules that achieve ionic gradients and underlie astrocyte signalling.
... This is further supported by a recent study which confirmed the functional importance of Type 1 IP 3 R function in mouse neuronal cells (van de Leemput et al. 2007). The existence of SOCE in neuronal cells was originally demonstrated in bovine adrenal chromaffin cells and in the PC12 cell line (Cheek and Thastrup 1989;Clementi et al. 1992) and later in other neuronal cells including the Aplyesia bag cell neurons (Philipp et al. 1998;Pizzo et al. 2001). Importantly, the relevance of SOCE channels in the development and plasticity of the nervous system was initially demonstrated by Mu-ming Poo's group (Nishiyama et al. 2000). ...
Calcium (Ca²⁺) is a ubiquitous second messenger that performs significant physiological task such as neurosecretion, exocytosis, neuronal growth/differentiation, and the development and/or maintenance of neural circuits. An important regulatory aspect of neuronal Ca²⁺ homeostasis is store-operated Ca²⁺ entry (SOCE) which, in recent years, has gained much attention for influencing a variety of nerve cell responses. Essentially, activation of SOCE ensues following the activation of the plasma membrane (PM) store-operated Ca²⁺ channels (SOCC) triggered by the depletion of endoplasmic reticulum (ER) Ca²⁺ stores. In addition to the TRPC (transient receptor potential canonical) and the Orai family of ion channels, STIM (stromal interacting molecule) proteins have been baptized as key molecular regulators of SOCE. Functional significance of the TRPC channels in neurons has been elaborately studied; however, information on Orai and STIM components of SOCE, although seems imminent, is currently limited. Importantly, perturbations in SOCE have been implicated in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of SOCC in neurodegeneration would presumably unveil avenues for plausible therapeutic interventions. We thus review the role of SOCE-regulated neuronal Ca²⁺ signaling in selecting neurodegenerative conditions.
... In this study we show that OAG-induced Ca 21 oscillations depend on the presence of TRPC3 in cultured astrocytes, that TRPC3 promotes astrogliosis and cortical edema in vivo, and these TRPC3 activities are attenuated by TRPC1, both in vitro and in vivo. . Asterisks assign the significant differences compared to the corresponding wt at the same hour (*p < 0.05, ** p < 0.01, *** p < 0.001) oscillations as well as ATP-, substance P-, and bradykinin-induced Ca 21 signals in cultured rat cortical and spinal cord astrocytes, in 1321N1 human astrocytoma cells and in primary mouse striatal and cultured cortical astrocytes were suggested to depend on TRPCs (Akita & Okada, 2011;Grimaldi et al., 2003;Miyano et al., 2010;Nakao et al., 2008;Shirakawa et al., 2010;Streifel et al., 2014;Streifel et al., 2013) which presumably are involved in Na 1 and Ca 21 influx including store-operated Ca 21 entry in these cells (Golovina, 2005;Pizzo, Burgo, Pozzan, & Fasolato, 2001;Reyes et al., 2013). Using the appropriate Trpc-deficient mice as controls we could show that neither TRPC3 nor TRPC1 significantly contribute to Ca 21 entry after store-depletion in cultured cortical astrocytes (Figure 2). ...
Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca(2+) . Transient receptor potential canonical (TRPC) channels may contribute to Ca(2+) influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca(2+) entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3(-/-) but increased in Trpc1(-/-) mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca(2+) signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.
... In contrast, experimental evidence indicates the role for TRP channels. They are expressed in astrocytes at both mRNA and protein levels, and TRP activity is involved in shaping astroglial Ca 2+ signals (Pizzo et al., 2001;Grimaldi et al., 2003;Golovina, 2005). Further analysis revealed that in astrocytes the TRP channels are assembles of brain native heteromultimers (Golovina, 2005;Malarkey et al., 2008) containing obligatory TRPC1 (channel forming subunit) and TRPC4 and/or TRPC5 (auxiliary subunits). ...
The name astroglia unifies many non-excitable neural cells that act as primary homeostatic cells in the
nervous system. Neuronal activity triggers multiple homeostatic responses of astroglia that include increase in metabolic activity and synthesis of neuronal preferred energy substrate lactate, clearance of neurotransmitters and buffering of extracellular K+ ions to name but a few. Many (if not all) of astroglial homeostatic responses are controlled by dynamic changes in the cytoplasmic concentration of two cations, Ca2+ and Na+. Intracellular concentration of these ions is tightly controlled by several transporters and can be rapidly affected by activation of respective fluxes through ionic channels or ion exchangers. Here we provide a comprehensive review of astroglial Ca2+ and Na+ signalling.
... In separate experiments, astrocytes in the cultures were identified on the basis of immunocytochemical staining with glial fibrillary acidic protein (GFAP) and[Ca 2+ ]i responses to direct glutamate stimulation (data not shown). We found that astrocytes demonstrated[Ca 2+ ]i responses to glutamate consistently only in the high micromolar and low millimolar range, which is consistent with a number of previous reports[11][12][13]. The ROIs were then used to measure fluorescence intensity over time in the specified area and created a data set based on ROIs. ...
... In separate experiments, astrocytes in the cultures were identified on the basis of immunocytochemical staining with glial fibrillary acidic protein (GFAP) and [Ca 2+ ] i responses to direct glutamate stimulation (data not shown). We found that astrocytes demonstrated [Ca 2+ ] i responses to glutamate consistently only in the high micromolar and low millimolar range, which is consistent with a number of previous reports [11][12][13]. The ROIs were then used to measure fluorescence intensity over time in the specified area and created a data set based on ROIs. ...
Glutamate (GLU) binding to neurons can cause dynamic changes in intracellular calcium. We tested effects of a 3-group submaximal glutamate stimulus (250, 500 and 750 nanomolar GLU in randomized orders) on neurons in culture, and measured intracellular calcium dynamics in cultures high and low in glia at 8 and 9 days in vitro. Gliadepleted cultures responded to increasing GLU with synchronized dynamics, leading to a greater “area under the curve” (AUC) for intracellular calcium over time. The AUC determined if the neuron would respond dynamically to the next addition of glutamate. This observation was not displayed within cultures high in glia, where AUC returned to baseline with every GLU addition, regardless of order of addition. Furthermore, the 3-group stimulus resulted in decreasing average AUC, regardless of order. In contrast, for cultures depleted of glia, the deciding factor of a responding cell to dynamically respond to GLU additions depended on the ability of the cell to distribute the calcium load (AUC) of the prior addition. Determining how neurons respond and behave such as in the presence of functional or dysfunctional glia, may help our understanding of signal processing in the brain.
... This process is termed capacitive Ca entry (CCE) or store operated Ca entry (SOCE), and probably is mediated by heteromeric TRPC1, TRPC4, and TRPC5 channels. Glutamate receptor activation or depletion of internal stores lead to [Ca ] increase and Ca oscillations in cultured astrocytes, which are blocked by La and RT-PCR identified transcripts encoding TRPC1, 3, 4, and 6 in these cells (Pizzo et al. 2001). In cultured astrocytes, SOCE is confined to microdomains in the plasma membrane that are located in close proximity to the endoplasmic reticulum and involve TRPC1, as shown by an antisense technique (Golovina 2005). ...
This resource is the long-awaited new revision of the most highly regarded reference volume on glial cells, and has been completely revised, greatly enlarged, and enhanced with full color figures throughout. Neglected in research for years, it is now evident that the brain only functions in a concerted action of all the cells, namely glia and neurons. Seventy one chapters comprehensively discuss virtually every aspect of normal glial cell anatomy, physiology, biochemistry and function, and consider the central roles of these cells in neurological diseases including stroke, Alzheimer disease, multiple sclerosis, Parkinson's disease, neuropathy, and psychiatric conditions. With more than 20 new chapters it addresses the massive growth of knowledge about the basic biology of glia and the sophisticated manner in which they partner with neurons in the course of normal brain function.
... Expression of Orai/STIM1 proteins was detected in the astroglial cell line U373 MG [63] and in primary cultured astrocytes [64]. At the same time, there is ample evidence for expression of TRP channels in astroglia, and these channels were found to contribute to Ca 2+ signalling in astroglia [65][66][67][68]. The main type of store-operated channel is represented by TRPC1/4/5 heteromers and inhibition of their function and/or expression suppresses SOCE in astroglia [65; 69]. ...
Pathological remodelling of astroglia represents an important component of the pathogenesis of Alzheimer's disease (AD). In AD astrocytes undergo both atrophy and reactivity; which may be specific for different stages of the disease evolution. Astroglial reactivity represents the generic defensive mechanism, and inhibition of astrogliotic response exacerbates b-amyloid pathology associated with AD. In animal models of AD astroglial reactivity is different in different brain regions, and the deficits of reactive response observed in entorhinal and prefrontal cortices may be linked to their vulnerability to AD progression. Reactive astrogliosis is linked to astroglial Ca2+ signalling, this latter being widely regarded as a mechanism of astroglial excitability. The AD pathology evolving in animal models as well as acute or chronic exposure to -amyloid induce pathological remodelling of Ca2+ signalling toolkit in astrocytes. This remodelling modifies astroglial Ca2+ signalling and may be linked to cellular mechanisms of AD pathogenesis.
... For example, in native HEK293 cells, Trp1, Trp2, Trp4, and Trp6 are expressed, but no diacylglycerol-sensitive Ca 2ϩ entry, a property of Trp6, was noted [33]. A recent comparison notes similar properties of capacitative Ca 2ϩ currents in several cell lines, even though the cells expressed different Trp isoforms [40]. On the other hand, antisense Trp4 significantly inhibited endogenous capacitative Ca 2ϩ entry in murine L cells [13] and in SBAC cells expressing bTrp1 and bTrp4 [26]. ...
Capacitative Ca²⁺ entry plays a role in thapsigargin- and oxytocin-mediated increases in intracellular free Ca²⁺ in human myometrium. Members of the Trp protein family have been implicated in capacitative Ca²⁺ entry in a number of tissues. Pregnant human myometrium and the human myometrial cell line PHM1-41 expressed mRNA for hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7. A number of known splice variants of hTrp1 and hTrp4 were expressed in these cells. In addition, novel splice variants for hTrp1 and hTrp3 were discovered. hTrp1γ1 and hTrp1γ2 contain insertions between previously described exons 9 and 10 that would alter reading frame and produce Trp proteins truncated in the membrane spanning region if expressed. The hTrp3 variant introduces sequence between exons 8 and 9 that would insert 16 amino acids in the C-terminal region of the protein upstream of the calmodulin and inositol 1,4,5-triphosphate receptor interaction domain. hTrp1, hTrp3, and hTrp4 proteins were detected in both pregnant human myometrial and PHM1-41 membranes; a weak band consistent with hTrp6 expression was detected in pregnant human myometrium. These data are consistent with the presence of proteins that could form putative capacitative Ca²⁺ channels in human myometrium. Control of the activity of these channels may be important for the control of uterine contractile activity.
... Astrocytes express TRPC1,4,5 subunits at both mRNA and protein levels (Pizzo et al. 2001;Grimaldi et al. 2003;Golovina 2005;Malarkey et al. 2008). In TRPC heteromultimers the TRPC1 channel is obligatory subunit, whereas TRPC4 and TRPC5 proteins have an auxiliary role (Strubing et al. 2001;Hofmann et al. 2002). ...
Abstract Excitability of astrocytes is based on spatio-temporally organized fluctuations
of intracellular concentrations of two ions, Ca2+ and Na+. This is dictated by
ionic movements between intracellular compartments, and between the cytosol and
the extracellular space, achieved by concentration-driven diffusion through membrane
channels or transport by pumps and exchangers. Neuronal activity triggers
transient elevation of Ca2+ and Na+ in astrocytes; changes in cytosolic levels of
these ions translate into functional responses through multiple molecular cascades.
Aberrant ionic signaling contributes to pathological reactions of astroglia in various
forms of neurological diseases, such as stroke, epilepsy, and various neurodegenerative
and neuropsychiatric disorders.
... After the spontaneous second peak in [Ca 2+ ] i occurs, however, most cells appear to enter a post-tetanic phase of rhythmic [Ca 2+ ] i oscillations. It is likely that the [Ca 2+ ] i oscillations are heavily-dependent on calcium-induced calcium release (CICR) from intracellular stores [26,27,68,69] and/or sustained extracellular calcium influx through store-operated calcium (SOC) channels that act to replenish calcium stores [70,71]. [Ca 2+ ] i oscillations may also be functionally important in the bi-directional communication between neurons and astrocytes that accompanies TBS-induced changes in hippocampal synaptic plasticity [72,73]. ...
Over four decades ago, it was discovered that high-frequency stimulation of the dentate gyrus induces long-term potentiation (LTP) of synaptic transmission. LTP is believed to underlie how we process and code external stimuli before converting it to salient information that we store as 'memories'. It has been shown that rats performing spatial learning tasks display theta-frequency (3-12 Hz) hippocampal neural activity. Moreover, administering theta-burst stimulation (TBS) to hippocampal slices can induce LTP. TBS triggers a sustained rise in intracellular calcium [Ca2+]i in neurons leading to new protein synthesis important for LTP maintenance. In this study, we measured TBS-induced [Ca2+]i oscillations in thousands of cells at increasing distances from the source of stimulation. Following TBS, a calcium wave propagates radially with an average speed of 5.2 µm/s and triggers multiple and regular [Ca2+]i oscillations in the hippocampus. Interestingly, the number and frequency of [Ca2+]i fluctuations post-TBS increased with respect to distance from the electrode. During the post-tetanic phase, 18% of cells exhibited 3 peaks in [Ca2+]i with a frequency of 17 mHz, whereas 2.3% of cells distributed further from the electrode displayed 8 [Ca2+]i oscillations at 33 mHz. We suggest that these observed [Ca2+]i oscillations could lead to activation of transcription factors involved in synaptic plasticity. In particular, the transcription factor, NF-κB, has been implicated in memory formation and is up-regulated after LTP induction. We measured increased activation of NF-κB 30 min post-TBS in CA1 pyramidal cells and also observed similar temporal up-regulation of NF-κB levels in CA1 neurons following water maze training in rats. Therefore, TBS of hippocampal slice cultures in vitro can mimic the cell type-specific up-regulations in activated NF-κB following spatial learning in vivo. This indicates that TBS may induce similar transcriptional changes to spatial learning and that TBS-triggered [Ca2+]i oscillations could activate memory-associated gene expression.
... TRPC1 is a nonselective cation channel with equal permeability for Ca 2+ and Na + and is different from other TRPC channels some of which are highly Ca 2+ permeable with P Ca /P monovalent between 2 and 9 [7]. Astroglial cells express several types of TRPC proteins, which, through heteromeric assembly with the TRPC1 subunit (that is known to be obligatory for channel formation), form functional channels [8][9][10][11]. TRPC channels in astrocytes are responsible for generation of storeoperated Ca + entry (SOCE) although the mechanism linking them to the endoplasmic reticulum (ER) Ca 2+ store remains elusive. Targeting the TRPC1 subunit in astrocytes with a blocking antibody or by reducing TRPC1 expression with a silencing RNA treatment substantially reduced the SOCE in cultured astroglial cells [8,10]. ...
... Nonetheless, these channels are potentially important for controlling cytosolic sodium concentration because negative resting potential of astrocytes makes Na + virtually the sole permeating cation. Among many cationic channels, the products of TRP genes have been identi fi ed in astrocytes (Golovina 2005 ; Grimaldi et al. 2003 ; Malarkey et al. 2008 ; Pizzo et al. 2001 ) . These TRP channels are reported to be activated following intracellular Ca 2+ release acting as store-operated channels (Parpura et al. 2011 ) . ...
Astrocytes exhibit their excitability based on variations in cytosolic Ca(2+) levels, which leads to variety of signalling events. Only recently, however, intracellular fluctuations of more abundant cation Na(+) are brought in the limelight of glial signalling. Indeed, astrocytes possess several plasmalemmal molecular entities that allow rapid transport of Na(+) across the plasma membrane: (1) ionotropic receptors, (2) canonical transient receptor potential cation channels, (3) neurotransmitter transporters and (4) sodium-calcium exchanger. Concerted action of these molecules in controlling cytosolic Na(+) may complement Ca(2+) signalling to provide basis for complex bidirectional astrocyte-neurone communication at the tripartite synapse.
... This story provided valuable insights into possible novel roles of TRPA1 channels in a cell type where the expression and function of TRP channels (Pizzo et al. 2001;Song et al. 2005;Shibasaki et al. 2007), and in particular of TRPA channels (Golovina, 2005), had not been clearly resolved. Additionally, it drew attention to a number of potential mechanisms that, unexpectedly, could allow astrocytes to finely tune the activity of specific sets of synapses, rather than non-specifically modulating the activity of large groups of cells, as generally thought. ...
TRP channels were first identified as membrane proteins mediating phototransduction in fruit flies (Cosens & Manning, 1969; Montell & Rubin, 1989). Astrocytes were initially referred to as the silent elements of the nervous system (Kuffler et al., 1966; Ransom & Goldring, 1973). At the time these discoveries were made, few would have suspected TRP channels and astrocytes could contribute significantly to our understanding of brain signaling. Recent findings, however, put TRP channels and astrocytes in the spotlight, describe their ability to modulate the activity of specific sets of synapses, and raise some interesting questions. What makes astrocytes capable of exerting cell-specific effects on inter-neuronal signals? How do different synapses respond to changes in astrocytic function and in the local micro-structure of the neuropil? Can astrocytes be considered good candidate targets for therapeutic intervention to treat neurological diseases? Here I discuss the recent developments on TRP channels and astrocytes that have made us aware of the many structural and functional features of synapses that still need to be discovered and that could lead a new avant-garde in decoding the cellular and molecular basis of brain (dys)function.
Calcium signaling is probably one of the evolutionary oldest and the most common way by which the signal can be transmitted from the cell environment to the cytoplasmic calcium binding effectors. Calcium signal is fast and due to diversity of calcium binding proteins it may have a very broad effect on cell behavior. Being a crucial player in neuronal transmission it is also very important for glia physiology. It is responsible for the cross-talk between neurons and astrocytes, for microglia activation and motility. Changes in calcium signaling are also crucial for the behavior of transformed glioma cells. The present chapter summarizes molecular mechanisms of calcium signal formation present in glial cells with a strong emphasis on extracellular nucleotide-evoked signaling pathways. Some aspects of glioma C6 signaling such as the cross-talk between P2Y1 and P2Y12 nucleotide receptors in calcium signal generation will be discussed in-depth, to show complexity of machinery engaged in formation of this signal. Moreover, possible mechanisms of modulation of the calcium signal in diverse environments there will be presented herein. Finally, the possible role of calcium signal in glioma motility is also discussed. This is a very important issue, since glioma cells, contrary to the vast majority of neoplastic cells, cannot spread in the body with the bloodstream and, at least in early stages of tumor development, may expand only by means of sheer motility.
Astrocytes are neural cells of ectodermal, neuroepithelial origin that provide for homeostasis and defense of the central nervous system (CNS). Astrocytes are highly heterogeneous in morphological appearance; they express a multitude of receptors, channels, and membrane transporters. This complement underlies their remarkable adaptive plasticity that defines the functional maintenance of the CNS in development and aging. Astrocytes are tightly integrated into neural networks and act within the context of neural tissue; astrocytes control homeostasis of the CNS at all levels of organization from molecular to the whole organ.
Alzheimer’s disease (AD) is the most common neurodegenerative disease in the world. The “amyloid hypothesis” is one of the predominant hypotheses for the pathogenesis of AD. Besides, tau protein accumulation, calcium homeostasis disruption, and glial cell activation are also remarkable features in AD. Recently, there are some reports showing that TRPC channels may function in AD development, especially TRPC6. In this chapter, we will discuss the evidence for the involvement of TRPC channels in Alzheimer’s disease and the potential of therapeutics for AD based on TRPC channels.
Neuronal exocytotic release of glutamate at synapses involves a highly specialized vesicular apparatus, consisting of a variety of proteins connected to the vesicles or required for vesicular fusion to the presynaptic membrane. Astrocytes also release glutamate, and recent evidence indicates that this release can modify neuronal function. Several mechanisms have been proposed for astrocytic release of glutamate under pathological conditions, such as reversal of glutamate transporters and opening of volume sensitive ion channels. In this review we limit our discussion to findings supporting the exocytotic release of glutamate, as well as two new pathways implicated in this release, the ionotropic (P2X) purinergic receptors and gap junction hemichannels.
Objectives:
Transient receptor potential canonical type 3 (TRPC3) channels are activated in B lymphoblast cell lines from patients with bipolar disorder (BD), and its expression is reduced by chronic lithium treatment, implicating TRPC3 in the intracellular calcium (Ca(2+) ) dyshomeostasis of BD. Thrombin, via a protease-activated receptor, moderates Ca(2+) signaling and TRPC3 in astrocytes, and also cell proliferation. We examined whether lithium pretreatment attenuates thrombin-stimulated TRPC3 expression and function in astrocytes, and levels of the calcium-binding peptide, S100B, which is expressed mainly in these cells.
Methods:
Human astroglioma, U-87MG, cells were pretreated with 1 mmol L(-1) LiCl for 1 day (acute), 3 days (subacute), and 7 days (chronic). To examine the role of TRPC3, genetically stable knockdown TRPC3 cells (TRPC3(Low) cells) were constructed using U-87MG cells. Thrombin (2.0 U/mL)-stimulated Ca(2+) mobilization was measured by ratiometric fluorimetry. Changes in TRPC3 and S100B expression levels were determined by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. Cell proliferation was also measured using the WST-8 assay.
Results:
In this cell model, thrombin-stimulated Ca(2+) mobilization, and both TRPC3 and S100B expression were suppressed by chronic LiCl pretreatment and the knockdown of TRPC3. Additionally, cell proliferation was attenuated in TRPC3(Low) cells, compared with the negative control vector-transfected cell.
Conclusions:
The reduced Ca(2+) mobilization and S100B expression levels following chronic LiCl pretreatment and in TRPC3(Low) cells support the notion that TRPC3 modulates S100B expression and is the target of the LiCl effect. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological intracellular Ca(2+) disturbances as observed in BD, accounting, in part, for its mood-stabilizing effects.
The expression of voltage-gated Ca2+ channels on astrocytes in culture has already been shown 20 years ago (MacVicar, 1984). A detailed investigation has recently identified a large variety of subtypes present in cultured cells, both on the protein and mRNA level, namely α1B (N-type), α1c (L-type), α1D (L-type), α1E (R-type), and α1G (T-type), but not α1A (P/Q-type) channels (Latour et al., 2003). The expression of Ca channels in astrocytes in situ is controversial. There was no evidence found for Ca2+ channels in astrocytes in acute slices from the visual cortex or the CA1 hippocampal region of developing rats and the depolarization-induced [Ca2+]i increases in astrocytes was solely attributed to the activation of metabotropic receptors by neurotransmitters, such as glutamate, released by synaptic terminals upon depolarization (Carmignoto et al., 1998). In contrast, freshly isolated astrocytes from 2–6 week old rat hippocampi showed verapamil-sensitive increases in Ca2+ due to depolarization by high K+ supporting the presence of voltage-gated Ca2+ channels (Fraser et al., 1995). For more details on Ca2+ channels see also Chapter 7.
Functional plasticity is an important property of the central nervous system (CNS). A major site for this plasticity is at the chemical synapse. Here, neurons and astrocytes intermingle forming a morphologically intimate relationship (Chapters 3 and 4) where astrocytes are favorably positioned to exchange signals with neurons (Figure 15.1). Ca2+ entry though voltage-gated channels into the presynaptic terminal signals to the secretory machinery, which allows the release of neurotransmitter stored in synaptic vesicles into the synaptic cleft. Released neurotransmitter then signals to the postsynaptic neuron by activating its receptors (Figure 15.1, arrow 1). Under certain circumstances, neurotransmitter can ‘spillover’ from the synaptic cleft and reach neurotransmitter receptors in adjacent astrocytes (Figure 15.1, arrow 2), eliciting astrocytic increases in intracellular Ca2+ ion concentration [Ca2+]i. These evoked and/or spontaneous elevations of [Ca2+]i in astrocytes can cause the release of a neurotransmitter, e.g., glutamate, from astrocytes, which signals to the presynaptic nerve terminal to modulate synaptic neurotransmission and/or released glutamate can affect postsynaptic cells (Figure 15.1, arrows 3 and 4). Additional signaling can occur between astrocytes in the form of intercellular Ca2+ waves (Figure 15.1, arrows 5; Chapters 12-14). In this chapter the focus is on glutamate-mediated bi-directional signaling between neurons and astrocytes.
The cell types comprising brain cellular network include neurons, astrocytes, and capillary microvasculature. Astrocytes extend foot processes and make close contact with neuronal cells and form a continuous layer to ensheath the outer surface of the capillaries and small arterioles to sense neuronal activity and transduce the signal to the cerebral microvessels. The mechanisms through which these three cell types interact to regulate brain function remains obscure. Activated neurons release adenosine that stimulates release of vasoactive factors including reactive oxygen species to induce vasodilatation. The discovery that brain cell types express cytochrome (CYP) ω-hydroxylases and CYP epoxygenases that catalyze carboxylation and epoxidation of the substrate arachidonic acid (AA) to form the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) and the vasodilatory regioisomeric epoxyeicosatrienoic acids (EETs), respectively, advances our knowledge of the mechanisms through which the dynamics of cerebral blood flow are regulated under normal or stress conditions. Both 20-HETE and the EETs target the KCa channel to elicit opposing biological actions. 20-HETE mediates pressure-evoked myogenic cerebral autoregulation, whereas the EETs induce cerebral vasodilatation and couple neuronal activity to hyperemic cerebral blood flow. In addition to their anti-inflammatory, neuroprotection, and vasodilatory actions, the EETs also posses mitogenic and angiogenic properties and promote vessel growth that could be targeted for their anti-tumorigenic and neuroprotective actions. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
This chapter focuses on astrocytes, a subtype of glial cell, which exhibit a form of excitability based on intracellular Ca2+ elevations that can stimulate glutamate release from astrocytes. The mechanism underlying this release is exocytosis. Astrocytes express the protein components of the exocytic-secretory machinery, including the core fusion complex as well as the transporters and pumps necessary for filling astrocytic vesicles with glutamate. The characteristics of exocytosis in astrocytes are distinct from those observed in neurons because of differences in the expression of the subtypes of exocytic protein subtypes. The morphological arrangements of the exocytic secretory machinery and the functional neurotransmitter receptors in astrocytic processes enable them to receive signals, focally, from adjacent synaptic terminals and respond back to terminals/dendrites via exocytic glutamate release. Astrocytes have the potential to play an active role in the computational power of the brain.
Astrocytes are organized in networks via gap junction channels constituted by connexin (Cx) 30 and Cx43. Since we observed that the mRNA expression of Cx30, but not Cx43, was enhanced after sleep deprivations (SD) in the mouse cortex and hippocampus, the goal of my thesis was to investigate whether and how Cx30 is involved in sleep homeostasis. First, I investigated the effects of sleep/wake-affecting molecules on gap junctional communication (GJC) of astrocytes in acute slices of the mouse cortex. We found that modafinil, a wakefulness-promoting drug, enhanced astroglial GJC, whereas γ-Hydroxybutyric acid (GHB), a sleep-promoting agent, and two general anesthetics, propofol and ketamine, decreased GJC, suggesting that astroglial networks are bidirectionally regulated by sleep/wake-affecting drugs. Then I addressed the role of Cx30 using Cx30 knockout (KO) mice. Compared to wild type (WT) mice, Cx30 KO exhibited a deficit in maintaining wakefulness during periods of high sleep pressure: they needed more stimuli to be maintained awake during gentle SD and they exhibited an increase in slow wave sleep during instrumental SD. To probe the possible causes of the phenotype, we found that: 1) astroglial GJC was enhanced in WT mice after SD, and such enhancement depended on both neuronal activity and the presence of Cx30; 2) mRNA levels of several genes involved in brain energy metabolism were decreased in multiple brain structures of the Cx30 KO. In summary, these results suggest that astroglial Cx30 plays an important role in sleep homeostasis, possibly by enhancing astroglial metabolic functions to fulfil the high energy demand during periods of elevated sleep pressure.
Calcium (Ca2+), as a ubiquitous second messenger, performs significant physiological tasks in regulating a plethora of neuronal functions including neurosecretion, exocytosis, neuronal growth/differentiation, and the development and maintenance of neural circuits. An important regulatory aspect of neuronal Ca2+ homeostasis is store-operated Ca2+ entry (SOCE), which, in recent years, has gained much attention for influencing a variety of nerve cell responses. Essentially, activation of SOCE ensues following the activation of the plasma membrane (PM) store-operated Ca2+ channels (SOCC) triggered by the depletion of endoplasmic reticulum (ER) Ca2+ stores. In addition to the TRP (Transient receptor potential) family of ion channels, the recently identified Orai and STIM (stromal interacting molecule) proteins have been baptized as key molecular components of SOCE. Functional significance of the TRP channels in neurons has been elaborately studied however, information on Orai and STIM components of SOCE, although seems imminent, is currently limited. Importantly, perturbations in SOCE have been implicated in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of SOCC in neurodegeneration would presumably unveil avenues for plausible therapeutic interventions. We thus review the role of SOCE-regulated neuronal Ca2+ signaling in select neurodegenerative conditions.
The brain possesses two intricate mechanisms that fulfill its continuous metabolic needs: cerebral autoregulation, which ensures constant cerebral blood flow over a wide range of arterial pressures and functional hyperemia, which ensures rapid delivery of oxygen and glucose to active neurons. Over the past decade, a number of important studies have identified astrocytes as key intermediaries in neurovascular coupling (NVC), the mechanism by which active neurons signal blood vessels to change their diameter. Activity-dependent increases in astrocytic Ca(2+) activity are thought to contribute to the release of vasoactive substances that facilitate arteriole vasodilation. A number of vasoactive signals have been identified and their role on vessel caliber assessed both in vitro and in vivo. In this review, we discuss mechanisms implicating astrocytes in NVC-mediated vascular responses, limitations encountered as a result of the challenges in maintaining all the constituents of the neurovascular unit intact and deliberate current controversial findings disputing a main role for astrocytes in NVC. Finally, we briefly discuss the potential role of pericytes and microglia in NVC-mediated processes.
Copyright © 2015. Published by Elsevier Ltd.
Alterations in intracellular Ca2+ concentration ([Ca2+]i) play a crucial role in fundamental cellular events from transcriptional regulation to migration and proliferation. The transient receptor potential (TRP) channels contribute to changes in [Ca2+]i by providing or modulating Ca2+ entry pathways, as well as by releasing Ca2+ from intracellular stores. On the basis of sequence homology, the TRP family can be divided into seven subfamilies: the TRPC (“canonical”) family, the TRPV (“vanilloid”) family, the TRPM (“melastatin”) family, the TRPP (“polycystin”) family, the TRPML (“mucolipin”) family, the TRPA (“ankyrin”) family, and the TRPN (“NOMPC”) family. In this review, we focus on the physiology and pathophysiology of mammalian TRPC channels in the nervous system. Seven mammalian TRPC proteins (TRPC1–7) have been described and are widely distributed in the brain from early embryonic days till adulthood, with the exception of TRPC2. TRPC channels are nonselective Ca2+-permeable channels, which can be activated by G-protein-coupled receptors and receptor tyrosine kinases. These channels have been reported to act as essential cellular sensors in multiple processes during neuronal development, including neural stem cell proliferation and differentiation, neuronal survival, neurite outgrowth and axon path finding, and synaptogenesis. Not surprisingly, studies on these channels also provide new insights into underlying mechanisms of various neuropsychiatric disorders. The present review summarizes the expressions of all TRPC subtypes in different brain regions and different neural cell types, aiming to serve as a useful reference for future studies in this field. We also discuss the most updated evidence implicating involvement of TRPC channels in the generation of pathophysiological states in nervous system and their potentials as being promising targets for drug development.
Transient receptor potential (TRP) channels have been increasingly implicated in the pathological mechanisms of CNS disorders. TRP expression has been detected in neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells as well as in the cerebral vascular endothelium and smooth muscle. In stroke, TRPC3/4/6, TRPM2/4/7, and TRPV1/3/4 channels have been found to participate in ischemia-induced cell death, whereas other TRP channels, in particular those expressed in nonneuronal cells, have been less well studied. This review summarizes the current knowledge on the expression and functions of the TRP channels in various cell types in the brain and our current understanding of TRP channels in stroke pathophysiology. In an aging society, the occurrence of stroke is expected to increase steadily, and there is an urgent requirement to improve the current stroke management strategy. Therefore, elucidating the roles of TRP channels in stroke could shed light on the development of novel therapeutic strategies and ultimately improve stroke outcome. © 2014 Wiley Periodicals, Inc.
The name astroglia unifies many non-excitable neural cells that act as primary homeostatic cells in the nervous system. Neuronal activity triggers multiple homeostatic responses of astroglia that include increase in metabolic activity and synthesis of neuronal preferred energy substrate lactate, clearance of neurotransmitters and buffering of extracellular K+ ions to name but a few. Many (if not all) of astroglial homeostatic responses are controlled by dynamic changes in the cytoplasmic concentration of two cations, Ca2+ and Na+. Intracellular concentration of these ions is tightly controlled by several transporters and can be rapidly affected by the activation of respective fluxes through ionic channels or ion exchangers. Here, we provide a comprehensive review of astroglial Ca2+ and Na+ signalling.
The destruction of calcium homeostasis is an important factor leading to neurological diseases. Store-operated Ca(2+) (SOC) channels are essential for Ca(2+) homeostasis in many cell types. However, whether SOC channels are involved in astrocyte activation induced by lipopolysaccharide (LPS) still remains unknown. In this study, we used LPS as an exogenous stimulation to investigate the role of SOC channels in astrocyte activation. Using calcium imaging technology, we first found that SOC channels blockers, 1-[h-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) and 2-aminoethyldiphenyl borate (2-APB), inhibited LPS induced [Ca(2+)]i increase, which prompted us to speculate that SOC channels may be involved in LPS induced astrocyte activation. Further experiments confirmed our speculation shown as SOC channels blockers inhibited LPS induced astrocyte activation characterized as cell proliferation by MTS and BrdU assay, raise in glial fibrillary acidic protein expression by immunofluorescence and Western Blot and secretion of interleukin 6 (IL-6) and interleukin 1β (IL-1β) by ELISA. So, our studies showed that SOC channels are involved in LPS-induced astrocyte activation.
Neuroglial cells are homeostatic neural cells. Generally, they are electrically non-excitable and their activation is associated with the generation of complex intracellular Ca(2+) signals that define the "Ca(2+) excitability" of glia. in mammalian glial cells the major source of Ca(2+) for this excitability is the lumen of the endoplasmic reticulum (ER), which is ultimately (re)filled from the extracellular space. This occurs via store-operated Ca(2+) entry (SoCE) which is supported by a specific signaling system connecting the ER with plasmalemmal Ca(2+) entry. Here, emptying of the ER Ca(2+) store is necessary and sufficient for the activation of SOCE, and without Ca(2+) influx via SOCE the ER store cannot be refilled. The molecular arrangements underlying SOCE are relatively complex and include plasmalemmal channels, ER Ca(2+) sensors, such as stromal interaction molecule, and possibly ER Ca(2+) pumps (of the SERCA type). There are at least two sets of plasmalemmal channels mediating SOCE, the Ca(2+)-release activated channels, Orai, and transient receptor potential (TRP) channels. The molecular identity of neuroglial SOCE has not been yet identified unequivocally. However, it seems that Orai is predominantly expressed in microglia, whereas astrocytes and oligodendrocytes rely more on TRP channels to produce SoCE. in physiological conditions the SoCE pathway is instrumental for the sustained phase of the Ca(2+) signal observed following stimulation of metabotropic receptors on glial cells.
Calcium signaling is probably one of the evolutionary oldest and the most common way by which the signal can be transmitted from the cell environment to the cytoplasmic calcium binding effectors. Calcium signal is fast and due to diversity of calcium binding proteins it may have a very broad effect on cell behavior. Being a crucial player in neuronal transmission it is also very important for glia physiology. It is responsible for the cross-talk between neurons and astrocytes, for microglia activation and motility. Changes in calcium signaling are also crucial for the behavior of transformed glioma cells. The present Chapter summarizes molecular mechanisms of calcium signal formation present in glial cells with a strong emphasis on extracellular nucleotide-evoked signaling pathways. Some aspects of glioma C6 signaling such as the cross-talk between P2Y(1) and P2Y(12) nucleotide receptors in calcium signal generation will be discussed in-depth, to show complexity of machinery engaged in formation of this signal. Moreover, possible mechanisms of modulation of the calcium signal in diverse environments there will be presented herein. Finally, the possible role of calcium signal in glioma motility is also discussed. This is a very important issue, since glioma cells, contrary to the vast majority of neoplastic cells, cannot spread in the body with the bloodstream and, at least in early stages of tumor development, may expand only by means of sheer motility.
Astrocytes are the most numerous glial cells. They fulfill a wide variety of vital functions, being in essence the wardens and governors of brain homeostasis. Astrocytes are integrated into a syncytium, being thus able to exchange molecules, and produce long-range signaling in a form of propagating Ca2+ waves. Astroglial cells are potentially capable to express virtually all types of neurotransmitter receptors known so far. These receptors can be activated by synaptically released neurotransmitters, by "glio" transmitters or by molecules diffusing in the brain extracellular space (volume transmitters). This chapter provides a concise summary of the properties of the main types of neurotransmitter receptors operative in astroglial cells. © Springer Science+Business Media, LLC 2009. All rights reserved.
Glial–neuronal communication was studied by monitoring the effect of intercellular glial Ca ²⁺ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca ²⁺ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 ± 2%). Excitation occurred in another five neurons (mean increase, 27 ± 5%). Larger amplitude Ca ²⁺ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca ²⁺ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and d (−)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca ²⁺ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.
Characterization of mammalian homologues ofDrosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca2+ influx activated in response to stimulation of Gq protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue,
TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain,
spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features,
compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca2+ release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing
TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca2+ influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and
for Ca2+ release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca2+ release. In fact, TRP7 expression did not affect capacitative Ca2+ entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn2+ influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell
patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation
currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca2+ at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid revealed an essential role of intracellular Ca2+ in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new
member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent
cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced
cation currents in various native systems.
Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.
In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.
Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types.
A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.
Long-term changes of synaptic strength in the central nervous system are mediated by an increase of cytosolic calcium concentration ([Ca2+]i) following activation of excitatory neurotransmitter receptors. These phenomena, which represent a possible cellular basis for learning and memory processes in eukaryotes, are believed to be restricted to neurons. Here we provide evidence for a long-term change of the response elicited by the excitatory neurotransmitter glutamate in a non-neuronal cell population of the central nervous system, i.e. visual cortical astrocytes in culture. Stimulation with glutamate induces in astrocytes a regular pattern of [Ca2+]i oscillations. A second stimulation, after an interval ranging from 2 to 60 min, induces an oscillatory response characterized by an increased frequency. Induction of this change in the astrocyte response is abolished by a specific inhibitor of the nitric oxide synthase and recovers upon exogenous nitric oxide generation or addition of a permeant cGMP analogue. Local brief pulses of glutamate to individual astrocytes, at a rate of 1 Hz, also elicit [Ca2+]i oscillations whose frequency increases following a second series of pulses. The long-lasting modification in the [Ca2+]i oscillatory response induced by glutamate in astrocytes demonstrates that in the central nervous system cellular memory is not a unique feature of neurons.
Calcium entry in non-excitable cells occurs through calcium-selective currents activated secondarily to store depletion and/or through non-selective cation channels (e.g., receptor- or second-messenger-activated channels). The driving force for calcium influx can be modified by chloride or potassium channels, which set the membrane potential of cells. Together, these conductances determine the extent of calcium entry. Mast cells are an excellent model system for studying calcium influx, because calcium-release-activated calcium currents (ICRAC), second-messenger-activated non-selective currents and chloride currents are present in these cells. Whole-cell patch-clamp recordings were used to test the effects of the commonly used calcium entry blockers econazole and SK&F 96365, as well as the antiallergic and anti-inflammatory drugs tenidap, ketotifen and cromolyn on these channels. All tested drugs blocked the three different channel types with a similar order of magnitude (IC50 values ranging from micromolar to millimolar). Hence, these drugs cannot be used to discriminate between different calcium entry mechanisms.
Neurotransmitter released from neurons is known to signal to neighbouring neurons and glia. Here we demonstrate an additional signalling pathway in which glutamate is released from astrocytes and causes an NMDA (N-methyl-D-aspartate) receptor-mediated increase in neuronal calcium. Internal calcium was elevated and glutamate release stimulated by application of the neuroligand bradykinin to cultured astrocytes. Elevation of astrocyte internal calcium was also sufficient to induce glutamate release. To determine whether this released glutamate signals to neurons, we studied astrocyte-neuron co-cultures. Bradykinin significantly increased calcium levels in neurons co-cultured with astrocytes, but not in solitary neurons. The glutamate receptor antagonists D-2-amino-5-phosphonopentanoic acid and D-glutamylglycine prevented bradykinin-induced neuronal calcium elevation. When single astrocytes were directly stimulated to increase internal calcium and release glutamate, calcium levels of adjacent neurons were increased; this increase could be blocked by D-glutamylglycine. Thus, astrocytes regulate neuronal calcium levels through the calcium-dependent release of glutamate.
Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert-butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)-ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.
The rat basophilic cell line RBL-1 is known to express high levels of the Ca2+ current activated by store depletion, known as Ca2+ release-activated Ca2+ current (ICRAC), the main Ca2+ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca2+ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between ICRAC and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces ICRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of ICRAC, since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca2+ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADPbetaS and AMP, but not by GDP or GTP.
Current models for agonist-activated Ca2+ entry in nonexcitable cells focus on the capacitative mechanism where entry is activated as a downstream result of the sustained depletion of agonist-sensitive stores without any direct requirement for inositol phosphates. This mechanism has been shown to be important for the sustained Ca2+ signals seen in a variety of nonexcitable cells under conditions of maximal stimulation. In contrast, relatively little attention has been given to Ca2+ entry under more physiological levels of agonist where, for example, oscillating Ca2+ responses are common. In recent studies using cells from the exocrine avian nasal gland, we have shown that agonist-activated Ca2+ entry under these conditions demonstrates properties that are inconsistent with current versions of the capacitative model. We now report that activation of this novel noncapacitative Ca2+ entry is via a distinct signaling pathway involving an agonist-induced, phospholipase A2-mediated generation of arachidonic acid.
TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661-671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 microM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 microM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.
The spatial-temporal characteristics of intracellular calcium ([Ca2+]i) changes elicited in neurons and astrocytes by various types of stimuli were investigated by means of confocal fluorescent microscopy in acute rat brain slices loaded with the Ca2+ indicator indo-1. Neurons and astrocytes from the visual cortex and CA1 hippocampal region were identified in situ on the basis of their morphological, electrophysiological, and pharmacological features. We show here that stimulation of neuronal afferents triggered periodic [Ca2+]i oscillations in astrocytes. The frequency of these oscillations was under a dynamic control by neuronal activity as it changed according to the pattern of stimulation. After repetitive episodes of neuronal stimulation as well as repetitive stimulation with a metabotropic glutamate receptor agonist, astrocytes displayed a long-lasting increase in [Ca2+]i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca2+]i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca2+]i oscillations in astrocytes represent a highly plastic signaling system that underlies the reciprocal communication between neurons and astrocytes.
Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10-15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release-activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.
Glial cells respond to various electrical, mechanical, and chemical stimuli, including neurotransmitters, neuromodulators, and hormones, with an increase in intracellular Ca2+ concentration ([Ca2+]i). The increases exhibit a variety of temporal and spatial patterns. These [Ca2+]i responses result from the coordinated activity of a number of molecular cascades responsible for Ca2+ movement into or out of the cytoplasm either by way of the extracellular space or intracellular stores. Transplasmalemmal Ca2+ movements may be controlled by several types of voltage- and ligand-gated Ca(2+)-permeable channels as well as Ca2+ pumps and a Na+/Ca2+ exchanger. In addition, glial cells express various metabotropic receptors coupled to intracellular Ca2+ stores through the intracellular messenger inositol 1,4,5-triphosphate. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca2+ responses. Such agonist specificity may provide a means for intracellular and intercellular information coding. Calcium signals can traverse gap junctions between glial cells without decrement. These waves can serve as a substrate for integration of glial activity. By controlling gap junction conductance, Ca2+ waves may define the limits of functional glial networks. Neuronal activity can trigger [Ca2+]i signals in apposed glial cells, and moreover, there is some evidence that glial [Ca2+]i waves can affect neurons. Glial Ca2+ signaling can be regarded as a form of glial excitability.
Calcium ions play crucial roles in a large variety of cell functions. The recent proposal that changes in the intracellular calcium concentration ([Ca2+]i) in astrocytes underline a reciprocal communication system between neurons and astrocytes encourages the interest in the definition of the various components participating in this novel Ca2+ signaling system. We investigate here whether functional voltage-operated calcium channels (Ca2+ VOCs), which are clearly expressed in cultured astrocytes, participate in the regulation of [Ca2+]i also in astrocytes in situ. Depolarization with 40-60 mM K+ was used to analyze the activity of Ca2+ VOCs in Indo-1-loaded astrocytes in acute slices from the visual cortex and the CA1 hippocampal region of developing rats. We demonstrate here that the depolarization-induced [Ca2+]i increases in astrocytes are solely attributed to the activation of metabotropic receptors by neurotransmitters, such as glutamate, released by synaptic terminals on depolarization. In fact, (1) the K+-induced [Ca2+]i increases in astrocyte [Ca2+]i were potently reduced by alpha-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor competitive inhibitor; (2) after emptying intracellular Ca2+ stores with cyclopiazonic acid, none of the astrocytes displayed a [Ca2+]i increase on the depolarizing stimulus; and (3) after inhibiting neurotransmitter secretion in neurons by incubating the slices with tetanus neurotoxin, no [Ca2+]i increase on K+ stimulation was observed in astrocytes. Finally, patch-clamp whole-cell recordings from hippocampal astrocytes in acute brain slices failed to reveal any voltage-dependent calcium currents. On the basis of these results, the various roles proposed for astrocyte Ca2+ VOCs in the CNS should be reconsidered.
Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.
In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.
Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
The rat basophilic cell line RBL-1 is known to express high levels of the Ca2+ current activated by store depletion, known as Ca2+ release-activated Ca2+ current (I-CRAC), the main Ca2+ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca2+ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between I-CRAC and eel metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces I-CRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of I-CRAC since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca2+ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis, Moreover, the inhibition is specific for ADP and is partially mimicked by ADP beta S and AMP, but not by GDP or GTP.
1. Carbachol, phenylephrine and substance P were each capable of producing a transient release of K (86 Rb) from rat parotid slices in the absence of extracellular Ca. 2. Each of the agonists was also capable of producing "cross-receptor inactivation"; that is, if a transient response was elicited by any one of the agonists then no transient response could be obtained by either of the other two. 3. Removal of carbachol from muscarinic receptors with atropine did not reverse the cross-receptor inactivation of the substance P response unless Ca was added with substance P or unless Ca was present when atropine was added. 4. It is concluded that the K release response in the parotid gland is mediated by receptor-controlled Ca influx sites. These influx sites also bind Ca at a location inaccessible to EGTA and release the bound Ca upon receptor activation. 5. It is also conlcuded that all three receptors (muscarinic, alpha-adrenergic, and peptide) appear to regulate the same Ca influx sites.
The recent discovery that the neurotransmitter glutamate can trigger actively propagating Ca2+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca2+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca2+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca2+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca2+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 microns/s at 21 degrees C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized.
The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for
these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory
elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate
receptor--one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and
promoting surface-membrane calcium influx--appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free
calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent
cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system
within the brain.
Cytosolic calcium oscillations induced by a wide range of agonists, particularly those which stimulate phosphoinositide metabolism, are the result of a periodic release of stored calcium. The formation of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) seems to play an important role because it can initiate this periodic behaviour when injected or perfused into a variety of cells. A two pool model has been developed to explain how Ins(1,4, 5)P3 sets up these calcium oscillations. It is proposed that Ins(1,4,5)P3 acts through its specific receptor to create a constant influx of primer calcium (Ca2+p) made up of calcium released from the Ins(1,4,5)P3-sensitive pool (ISCS) together with an influx of external calcium. This Ca2+p fails to significantly elevate cytosolic calcium because it is rapidly sequestered by the Ins(1,4,5)P3-insensitive (IICS) stores of calcium distributed throughout the cytosol. Once the latter have filled, they are triggered to release their stored calcium through a process of calcium-induced calcium release to give a typical calcium spike (Ca2+s). In many cells, each Ca2+s begins at a discrete initiation site from which it then spreads through the cell as a wave. The two pool model can account for such waves if it is assumed that calcium released from one IICS diffused across to excite its neighbours thereby setting up a self-propagating wave based on calcium-induced calcium release.
A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.
1. The L-type Ca2+ current was recorded in guinea-pig ventricular myocytes by the patch clamp technique in the whole-cell configuration. The modification of the current by intracellular application of proteases was studied. 2. During the first phase of action, trypsin, an endopeptidase, increased the amplitude of Ca2+ current about 3-fold. 3. Thereafter, there was a drastic slowing of the inactivation time course of the enhanced Ca2+ current. The half-time of inactivation increased from a control value of about 25 ms to values larger than 200 ms. 4. Cell dialysis with carboxypeptidase A, an exopeptidase, also enlarged the amplitude of Ca2+ current, but did not affect the kinetics of Ca2+ current. Leuaminopeptidase did not modify the Ca2+ current. 5. The hypothesis that Ca2+ channels are affected by the protease is supported by the fact that alterations of the extracellular Na+ or K+ concentration did not influence the modification of the membrane current. Another argument for the involvement of Ca2+ channels is that the modified membrane current could be blocked by inorganic and organic Ca2+ channel blockers (e.g. 10 microM-Cd2+, 100 microM-La3+ or 1 microM-D600). 6. Although the actions of trypsin and maximal concentrations of isoprenaline on the amplitude of the Ca2+ current were not additive, the slowing of inactivation by trypsin occurred independently from beta-adrenergic stimulation. 7. The effect of trypsin on the Ca2+ current could not be blocked by intracellular 5'-adenylyl-imidodiphosphate (AMP-PNP) or Rp-adenosine 3'5'-monothionophosphate (Rp-cAMPS), both of which are known to suppress the cyclic AMP-dependent phosphorylation of the Ca2+ channel. 8. It was concluded that trypsin may directly modify the membrane protein which forms the Ca2+ channel. Since the increment in peak Ca2+ current resembled the action of cyclic AMP-dependent phosphorylation, it may be related to the removal of a 'chemical' inactivation gate which is normally controlled by phosphorylation. The slowing of the time course of Ca2+ current inactivation by trypsin could be due to a modification of the voltage-dependent inactivation gate. Alternatively, the endopeptidase might remove an internal Ca2+ binding site normally responsible for Ca2+-dependent inactivation.
The changes in cytosolic free calcium concentration [( Ca2+]i) were monitored (fura-2) in single, isolated, mouse pancreatic acinar cells stimulated by acetylcholine (ACh). Responses to ACh at concentrations between 10(-7) and 5 x 10(-7) M are marked by the appearance of regular, sinusoidal, oscillations in [Ca2+]i. At 37 degrees C the oscillations are transient, being seen only in the initial rising phase of the calcium signal. At 30 degrees C regular oscillations can be maintained throughout the period of ACh application. This study reports that release of intracellular calcium and influx of extracellular calcium are both involved in the generation of these oscillatory calcium signals.
The fluorescent Ca2+ indicator fura 2 was used to measure cytosolic free [Ca2+] ([Ca2+]i) in order to obtain information about relative rates of Ca2+ influx into parietal cells during treatment with carbachol (a cholinergic agonist) or thapsigargin (TG, a Ca(2+)-mobilizing agent) or during reloading of the internal Ca2+ stores. In Ca(2+)-containing solutions, carbachol-, TG-, and reloading-stimulated Ca2+ entry exhibited nearly identical sensitivity to La3+ [inhibition constant (Ki) approximately 10 microM] or low pH (pKi approximately 7.0). In experiments in which carbachol and TG were used, there was no additional increase in [Ca2+]i when TG was added to carbachol-treated cells or when carbachol was added to cells previously treated with TG. Thus it is likely that a single Ca2+ entry pathway serves a signaling function as well as a role in refilling the Ca2+ store during reloading. Because the Ca2+ pathway is exquisitely sensitive to pH and serosal pH increases during stimulant-induced H+ secretion (which is activated by increases in [Ca2+]i), this mechanism will exert positive feedback on parietal cells in the intact stomach. When parietal cells were pretreated with carbachol in Ca(2+)-free solutions, reloading was independent of pH and La3+, suggesting that Ca(2+)-containing solutions should be used to determine the properties of the influx pathway.
The effects of 1-[2-(4-fluorophenyl)cyclohexyl]-2-[4-(3-phenylalkyl)-piperazin -1-yl]- ethanol, LU52396, on a) Ca2+ influx across the plasma membrane and b) Ca2+ mobilization from intracellular rapidly-exchanging Ca2+ stores were investigated in HeLa cells and in isolated microsomal fractions derived from the cerebellum and the skeletal muscle. LU52396 was found to be a potent inhibitor (Ki of about 2 microM) of the Ca2+ influx activated by depletion of intracellular Ca2+ stores, a phenomenon referred to as store-dependent or capacitative Ca2+ influx. Such an effect, which was reversed by cell washing, was mediated neither by a depolarization of the cell, with decrease in the driving force for cation influx, nor by a change of the intracellular pH, and might therefore be due to a direct action of the drug on either the responsible channel in the plasma membrane or, less likely, on its regulatory mechanisms. Additional effects, i.e. inhibition of receptor-mediated Ca2+ influx, of Ca2+ release from intracellular stores via either inositol 1,4,5-trisphosphate or ryanodine receptors, and of Ca2+ reuptake into the stores via sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPases, were also induced by the drug, however at concentrations 20-fold or more than those effective on the store-dependent influx. To our knowledge LU52396 is the first pharmacological tool that is found to be addressed with some preference to the store-dependent Ca2+ influx. It promises, therefore, to be useful for the characterization of the process, the identification of the responsible channel and, possibly, also of the molecular mechanisms through which these channels operate.
VIP plays an integral role in both protein and fluid secretion in many exocrine glands. By employing the perforated patch-clamp whole-cell recording technique we investigated the effects of VIP on membrane potential and transmembrane currents in avian exocrine salt gland cells. Prior to application of VIP, salt gland cells had a resting membrane potential close to -45 mV. When challenged with VIP (1-100 nM) a sustained depolarization to ECl- was induced which was mimicked by the application of cell-permeable cAMP analogues or forskolin (1 microM). By employing the voltage-clamp recording configuration a sustained increase in current was observed with a reversal potential which approximated ECl-. Ionic substitution experiments confirmed that the current was a Cl- conductance which was inhibited by the Cl- channel blockers flufenamic acid and niflumic acid and by the inhibitory cAMP isomer, adenosine-3',5'-cyclic monophosphothioate, Rp-isomer. Based on this, and the fact that the kinetic properties of the Cl- current activated by VIP are similar to those activated by cAMP, we propose that VIP-receptor interaction results in the activation of a cAMP-dependent Cl- current.
In current models describing agonist-induced oscillations in [Ca2+]i, Ca2+ entry is generally assumed to have a simple sustaining role, replenishing Ca2+ lost from the cell and recharging intracellular Ca2+ stores. In cells from the avian nasal gland, a model exocrine cell, we show that inhibition of Ca2+ entry by La3+, SK&F 96365, or by membrane depolarization, rapidly blocks [Ca2+]i oscillations but does so without detectable depletion of agonist-sensitive Ca2+ stores. As the rate of Mn2+ quenching during [Ca2+]i oscillations is constant, Ca2+ entry is not directly contributing to the [Ca2+]i changes and, instead, appears to be involved in inducing the repetitive release of Ca2+ from internal stores. Together, these data contradict current models in that (i) at the low agonist concentrations where [Ca2+]i oscillations are seen, generated levels of Ins(1,4,5)P3 are themselves inadequate to result in a regenerative [Ca2+]i signal, and (ii) Ca2+ entry is necessary to actually drive the intrinsic oscillatory mechanism.
1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)
We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ > Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.
Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.
In endothelial cells, different types of Ca2+ conductances have been described, but none of them has been clearly identified as I
CRAC, the Ca2+ release-activated Ca2+ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate (InsP
3), Ca2+ ionophores and Ca2+ pump inhibitors activates a Ca2+-selective conductance in the presence of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La3+. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an I
CRAC-like current. At 0 mV holding potential the estimated current density is –0.1 and –0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na+ currents are detectable in CPAE and HEK cells following store depletion in Ca2+- and Mg2+-free medium. The current carried by Na+ ions is similarly blocked by micromolar La3+, is inwardly rectifying and has a positive reversal potential.
Ca2+ influx into secretory cells of the intact salivary gland of the blowfly Calliphora erythrocephala elicited by the agonist 5-hydroxytryptamine (5-HT) or the Ca2+ uptake inhibitor thapsigargin was studied by using Fura-2 and digital fluorescence imaging and by recordings of the transepithelial potential. Application of saturating [5-HT] in the absence of Ca2+ (Ca2+o) from the bathing saline did not affect the initial Ca2+ transient but greatly attenuated the subsequent sustained Ca2+ elevation observed in the presence of Ca2+o demonstrating that the latter component of the [Ca2+]i response is largely dependent on Ca2+ entry across the baso-lateral plasma membrane. La3+ or Gd3+ (10 microM) mimicked the effects of the withdrawal of Ca2+o. Experimental attempts temporally to uncouple 5-HT stimulation and Ca2+ influx by withdrawal of Ca2+o during agonist application revealed a second Ca2+ entry pathway. This pathway was insensitive to 10 microM La3+ and produced transient [Ca2+]i increases whose amplitudes were a function of the [5-HT] during the preceding stimulation and that were selectively suppressed by 50 microM SK&F 96365. Both (10 microM) La(3+)-insensitive [Ca2+]i transients and (10 microM) La3+ inhabitable tonic [Ca2+]i increases could be sequentially activated in the presence of 5-HT or thapsigargin (1 microM). These results indicate that Ca2+ store depletion by 5-HT or thapsigargin activates two distinct store-operated Ca2+ entry pathways, one of which supports tonic [Ca2+]i increases. The other is transiently activated, even under conditions that prohibit store refilling and does not significantly contribute to the [Ca2+]i responses evoked by saturating 5-HT concentrations.
Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.
Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).