Capacitative Ca Entry Research Articles

Characterization of a 1,25(OH)2‐vitamin D3‐responsive capacitative Ca2+ entry pathway in rat osteoblast‐like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis.

Calcium-sensitive adenylyl cyclase/aequorin chimeras as sensitive probes for discrete modes of elevation of cytosolic calcium

The fact that Ca 2+ -sensitive adenylyl cyclases respond to discrete, physiological elevations in cytosolic Ca 2+ ([Ca 2+ ] i ) provides a means for coordinating the actions of the two major second-messenger signaling systems. It turns out that the Ca 2+ -sensitive adenylyl cyclases are extremely discriminating in the source of the Ca 2+ to which they respond. In intact nonexcitable cells, they are regulated only by' the entry of Ca 2+ that is triggered by depletion of internal stores, so-called capacitative Ca 2+ entry (CCE). Store depletion can be triggered by hormones coupled to phosphoinositidase C, by inhibition of the Ca 2+ -reuptake pumps of the endoplasmic reticula (ER), or by ionophore insertion in the ER membranes. However, when CCE is triggered, it regulates Ca 2+ -sensitive adenylyl cyclases, whether these occur in the cells naturally or after heterologous expression. A major conceptual breakthrough came with the targeting of aequorin to discrete domains of the cell by placing an organelle-targeting sequence upstream (or downstream) of the aequorin-coding region. This allowed measurement of [Ca 2+ ] in the ER, nuclear, and mitochondrial environments. This chapter describes some of the issues involved in validating this and related constructs and their application.

Propofol Attenuates Capacitative Calcium Entry in Pulmonary Artery Smooth Muscle Cells

Depletion of intracellular Ca2+ stores results in capacitative Ca2+ entry (CCE) in pulmonary artery smooth muscle cells (PASMCs). The authors aimed to investigate the effects of propofol on CCE and to assess the extent to which protein kinase C (PKC) and tyrosine kinases mediate propofol-induced changes in CCE. Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration was measured using fura 2 in PASMCs using a dual-wavelength spectrofluorometer. Thapsigargin (1 microM), a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was activated when extracellular Ca2+ (2.2 mM) was restored. Thapsigargin caused a transient increase in intracellular Ca2+ concentration (182+/-11%). Restoring extracellular calcium (to induce CCE) resulted in a peak (246+/-12% of baseline) and a sustained (187+/-7% of baseline) increase in intracellular Ca2+ concentration. Propofol (1, 10, 100 microM) attenuated CCE in a dose-dependent manner (peak: 85+/-3, 70+/-4, 62+/-4%; sustained: 94+/-5, 80+/-5, 72+/-5% of control respectively). Tyrosine kinase inhibition (tyrphostin 23) attenuated CCE (peak: 67+/-4%; sustained: 74+/-5% of control), but the propofol-induced decrease in CCE was still apparent after tyrosine kinases inhibition. PKC activation (phorbol 12-myristate 13-acetate) attenuated CCE (peak: 48+/-1%; sustained: 53+/-3% of control), whereas PKC inhibition (bisindolylmaleimide) potentiated CCE (peak: 132+/-11%; sustained: 120+/-4% of control). Moreover, PKC inhibition abolished the propofol-induced attenuation of CCE. Tyrosine kinases activate and PKC inhibits CCE in PASMCs. Propofol attenuates CCE primarily via a PKC-dependent pathway. CCE should be considered a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.

Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells.

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation.

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells.

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.

Identifying the Ca ++ signalling sources activating chloride currents in Xenopus oocytes using ionomycin and thapsigargin

The calcium ionophore, ionomycin (IM), and the sarcoplasmic/endoplasmic reticulum (SER) calcium pump inhibitor, thapsigargin (TG), were used to study the roles of Ca ++ from different sources in regulating Ca ++-dependent Cl − currents in Xenopus oocytes. The Ca ++-dependent Cl − currents, Ic, were measured in voltage-clamped oocytes (Vc = −60 mV). In the presence of extracellular Ca ++, both TG (0.1 to 10 μM) and IM (0.1 to 10 μM) induce release of Ca ++ from SER and activated capacitative Ca ++ entry (CCE) across the plasma membrane leading to activation of both “fast” and “slow” Cl − currents. The fast Ic was produced by Ca ++ release from SER while Ca ++ entry across the plasma membrane activated the slow Ic. Intracellular application of the calcium buffer, BAPTA, blocked activation of the slow Ic due to Ca ++ entry via CCE pathways, but not via IM-mediated movement across the plasma membrane. It is concluded that predominantly Ca ++ release from stores regulates a fast Ic while Ca ++ entry through CCE pathways regulates a slow Ic. Further, the CCE and slow Ic pathways must be located in spatially separated compartments since BAPTA can effectively abolish the effects of Ca ++ entry via the CCE pathway, but not by the IM-mediated entry pathway.

Positive regulation of capacitative Ca2+entry by intracellular Ca2+in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+in the opening of capacitative Ca2+entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl–current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl–response was recorded at controlled cytosolic Ca2+concentrations. Intracellular perfusion with a Ca2+-free solution containing 10mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+concentration up to 300nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+concentration of 110nM. Blockade of endogenous Cl–channels with anion channel blocker isolated Ca2+current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+at higher sensitivity compared to oocyte-endogenous CCE channels.

EGF enhances Ca2 mobilization and capacitative Ca2 entry in mouse mammary epithelial cells

EGF enhances Ca2 mobilization and capacitative Ca2 entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca2 entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca2 entry in mouse mammary epithelial cells

Capacitative Ca2+ entry is graded with degree of intracellular Ca2+ store depletion in bovine vascular endothelial cells

1. In endothelial cells, release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores activates Ca2+ influx via the capacitative Ca2+ entry (CCE) pathway. In cultured bovine pulmonary artery endothelial cells, we investigated the relationship between intracellular Ca2+ store load and CCE activity, as well as the kinetics of CCE activation and deactivation, by simultaneously measuring changes in [Ca2+]i and unidirectional manganese (Mn2+) entry through the CCE pathway. 2. Submaximal concentrations of ATP caused quantal release of Ca2+ from the ER, resulting in a dose-dependent depletion of Ca2+ stores and acceleration of Mn2+ entry. Mn2+ entry rate, as a measure of CCE activity, was graded with the amount of released Ca2+. Maximal activation of CCE did not require complete store depletion. 3. Slow depletion of the ER by exposure to the ER Ca2+ pump inhibitor cyclopiazonic acid resulted in a delayed activation of CCE, revealing a temporal dissociation between release of Ca2+ from intracellular stores and activation of CCE. 4. During [Ca2+]i oscillations, at frequencies higher than 0.5 spikes min-1, each Ca2+ spike resulted in a progressive acceleration of CCE without leading to oscillations of Ca2+ entry. In contrast, low frequency [Ca2+]i oscillations were paralleled by transient CCE that was activated and deactivated with each Ca2+ spike, resulting in an oscillatory pattern of Ca2+ entry. 5. It is concluded that CCE is a rapidly activating process which is graded with store depletion and becomes fully activated before complete depletion. The duration of CCE activation correlates with the degree of store depletion and the time that is required to refill depleted stores. Overall, a mechanism of graded CCE prevents exhaustion of intracellular Ca2+ reserves and provides an efficient way to respond to variable degrees of intracellular store depletion.

Caffeine exerts a dual effect on capacitative calcium entry in Xenopus oocytes

Caffeine increases the amplitude of the Cl − currents evoked by capacitative Ca 2+ entry (CCE) on thapsigargin-treated Xenopus oocytes. The caffeine-induced potentiation of the CCE process appears to rest on two distinct and additive components. The first component involves the cAMP second messenger system since it can be mimicked by either IBMX perfusion or cAMP microinjection into the oocyte and inhibited by the PKA inhibitory peptide i-PKA. The second component, although activatory, is dynamically related to the caffeine-evoked inhibition of InsP 3-mediated Ca 2+ release and may arise from an interaction between caffeine and the InsP 3 receptor in the context of a conformational coupling between the InsP 3 receptor and the channels responsible for CCE.

Cell proliferation is associated with enhanced capacitative Ca(2+) entry in human arterial myocytes.

Depletion of Ca(2+) stores in the sarcoplasmic reticulum (SR) activates extracellular Ca(2+) influx via capacitative Ca(2+) entry (CCE). Here, CCE levels in proliferating and growth-arrested human pulmonary artery smooth muscle cells (PASMCs) were compared by digital imaging fluorescence microscopy. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in proliferating PASMCs was twofold higher than that in growth-arrested cells. Cyclopiazonic acid (CPA; 10 microM), which inhibits SR Ca(2+)-ATPase and depletes inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, transiently increased [Ca(2+)](cyt) in the absence of extracellular Ca(2+). The addition of 1.8 mM Ca(2+) to the extracellular solution in the presence of CPA induced large increases in [Ca(2+)](cyt), indicative of CCE. The CPA-induced SR Ca(2+) release in proliferating PASMCs was twofold higher than that in growth-arrested cells, whereas the transient rise of [Ca(2+)](cyt) due to CCE was fivefold greater in proliferating cells. CCE was insensitive to nifedipine but was significantly inhibited by 50 mM K(+), which reduces the driving force for Ca(2+) influx, and by 0.5 mM Ni(2+), a putative blocker of store-operated Ca(2+) channels. These data show that augmented CCE is associated with proliferation of human PASMCs and may be involved in stimulating and maintaining cell growth.

Stimulation of Drosophila TrpL by capacitative Ca2+ entry

Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+>Sr2+>> Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE.

Dynamic regulation of [Ca2+]iby plasma membrane Ca2+-ATPase and Na+/Ca2+exchange during capacitative Ca2+entry in bovine vascular endothelial cells

The dynamic regulation of Ca2+extrusion by the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]ielicited by activation of capacitative Ca2+entry (CCE) served to characterize kinetic parameters of these Ca2+extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca2+- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]ihas been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+removal system. The contribution by the low-affinity NCX to Ca2+clearance became apparent at [Ca2+]i>~150 nM under conditions of submaximal activation of the PMCA.

Mechanisms of Ca2+store depletion in single endothelial cells in a Ca2+-free environment

Depletion of agonist-sensitive Ca2+stores results in activation of capacitative Ca2+entry (CCE) in endothelial cells. The proportion of Ca2+stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca2+-free environment results in emptying of bradykinin-sensitive Ca2+stores (BsS) and activation of CCE. In a Ca2+-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca2+-free solution). This effect was prevented by inhibition of the Na+–Ca2+exchange but not by a blockade of ryanodine-sensitive Ca2+release (RsCR). In contrast to BsS, mitochondrial Ca2+content remained unchanged in the Ca2+-free environment. Remarkably, activity of CCE (monitored as Mn2+influx) did not increase after depletion of BsS in the Ca2+-free environment. In contrast to Mn2+influx, the effect of re-addition of Ca2+to elevate bulk Ca2+concentration ([Ca2+]b) decreased with the time the cells rested in Ca2+-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+conditions the effect of Ca2+on [Ca2+]bwas reduced while it did not change the time the cells rested in Ca2+-free solution. After a 2 min period in low Na+conditions, ryanodine-induced Ca2+extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+on [Ca2+]bin low Na+conditions. Collapsing subplasmalemmal Ca2+stores with nocodazole, increased the effect of Ca2+on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+on [Ca2+]bwas not reduced in Ca2+-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+pools that deplete rapidly in a Ca2+-free environment. Subplasmalemmal ryanodine-sensitive Ca2+stores (RsS) are emptied in Ca2+-free /low Na+solution and re-sequester Ca2+which enters the cells prior an increase in [Ca2+]boccurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.

Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel.

Capacitative Ca2+ entry (CCE) is Ca2+ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca2+ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca2+ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca2+-permeable cation channels activated by agonists that stimulate IP3 formation-very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca2+ entry channel. The role of Trp's in Ca2+ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of Ca2+ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca2+ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca2+ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca2+ channels.

Receptor-activated Ca2+ influx: capacitative Ca2+ entry and TRP proteins

Receptor-activated Ca2+ channels (RACC) are triggered in response to activation of G protein-coupled receptors or tyrosine kinase-coupled receptors. RACCs, together with voltage-dependent Ca2+ channels, form physiologically the most important Ca2+ influx pathways, being highly diverged in activation mechanisms and Ca2+ permeability. Characterization of mammalian homologues of Drosophila TRP proteins has been an important clue for understanding molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. Recent issues have been whether any members of the TRP family form capacitative Ca2+ entry (CCE) channels activated by release of Ca2+ from internal stores and their depletion. We have isolated cDNAs that encode seven mouse TRP homologues, TRP1-7. TRP homologues are distributed differently among tissues, although they are all abundant in the brain. Functional characterization of TRP proteins recombinantly expressed in HEK cells indicate that TRP5 is highly permeable to Ca2+, while TRP3 and 7 are non-selective cation channels. The results demonstrate that TRP3,5,7 are capable of generating Ca2+ currents after desensitization of the stimulated G-protein-coupled receptors and replenishment of stores, suggesting that store depletion is not necessary to maintain activity of the TRP homologues. Ca2+ positively regulates TRP channels through Ca(2+)-calmodulin pathways, but via different Ca(2+)-calmodulin-dependent enzymes. Thus, activation of TRP channels is not tightly coupled with store depletion as CCE, suggesting that CCE (or CRAC) channels are molecular entities separate from TRP.

Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells.

1. Intracellular Ca2+ ([Ca2+]i) signals were studied with spatial resolution in bovine vascular endothelial cells using the fluorescent Ca2+ indicator fluo-3 and confocal laser scanning microscopy. Single cells were stimulated with the purinergic receptor agonist ATP resulting in an increase of [Ca2+]i due to intracellular Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores. ATP-induced Ca2+ release was quantal, i.e. submaximal concentrations mobilized only a fraction of the intracellularly stored Ca2+. 2. Focal receptor stimulation in Ca2+-free solution by pressure application of agonist-containing solution through a fine glass micropipette resulted in a spatially restricted increase in [Ca2+]i. Ca2+ release was initiated at the site of stimulation and frequently propagated some tens of micrometres into non-stimulated regions. 3. Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). CCE was initially colocalized with Ca2+ release. Following repetitive focal stimulation, however, CCE became detectable at remote sites where no Ca2+ release had been observed. In addition, the rate of Ca2+ store depletion with repetitive local activation of release in Ca2+-free solution was markedly slower than that elicited by ATP stimulation of the entire cell. 4. From these experiments it is concluded that both intracellular IP3-dependent Ca2+ release and activation of CCE are controlled locally at the subcellular level. Moreover, redistribution of intracellular Ca2+ stored within the endoplasmic reticulum efficiently counteracts local store depletion and accounts for the spatial spread of CCE activation.

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca 2+] i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca 2+] i dose dependently with an EC 50 of ~ 0.15 μM. The Ca 2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca 2+ store with thapsigargin for 7 min abolished the [Ca 2+] i increases evoked by bradykinin. Removal of extracellular Ca 2+ reduced the thapsigargin response by ~50%. The Ca 2+ signal was initiated by Ca 2+ release from the internal store followed by capacitative Ca 2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La 3+ and Gd 3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca 2+ store for 30 min with another inhibitor of the internal Ca 2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca 2+] i, thus suggesting that the thapsigargin-evoked Ca 2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatemt with La 3+ (or Gd 3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca 2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na + had no effect. Collectively, our results imply that thapsigargin increased [Ca 2+] i in MDCK cells by depleting the internal Ca 2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca 2+ pump.