SOC Channels Research Articles

Calcium signaling mediated by P2Y receptors in mouse taste cells.

Evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the taste bud. Recently, we found that ATP, a ubiquitous neurotransmitter/neuromodulator, mobilizes intracellular Ca2+ in taste cells by activating P2Y receptors. Here, P2Y receptor-cellular response coupling was characterized in detail using single cell ratio photometry and the inhibitory analysis. The sequence of underlying events was shown to include ATP-dependent activation of PLC, IP3 production, and IP3 receptor-mediated Ca2+ release followed by Ca2+ influx. Data obtained favor SOC channels rather than receptor-operated channels as a pathway for Ca2+ influx that accompanies Ca2+ release. Intracellular Ca2+ mobilized by ATP is apparently extruded by the plasma membrane Ca2+-ATPase, while a contribution of the Na+/Ca2+ exchange and other mechanisms of Ca2+ clearance is negligible. Cyclic AMP-dependent phosphorylation is likely to control a gain of the phosphoinositide cascade involved in ATP transduction. ATP-responsive taste cells are abundant in circumvallate, foliate, and fungiform papillae. Taken together, our observations point to a putative role for ATP as a neurotransmitter operative in the taste bud.

A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes.

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.

Ca2+]i oscillations induced by high [K+]o in acetylcholine-stimulated rat submandibular acinar cells: regulation by depolarization, cAMP and pertussis toxin.

Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels

The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca 2+ signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP 3)-mediated Ca 2+ release and store-operated Ca 2+ entry (SOC). In addition to abrogating hormone-evoked Ca 2+ responses, 2-APB could antagonise Ca 2+ signals evoked by a membrane permeant InsP 3 ester. 2-APB also slowed the recovery of intracellular Ca 2+ signals consistent with an effect on Ca 2+ ATPases. The inhibitory action of 2-APB on InsP 3 receptors (InsP 3Rs), SOC channels and Ca 2+ pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca 2+ responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca 2+ channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca 2+ signalling in electrically-stimulated cardiac myocytes, where InsP 3Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca 2+ signalling in cell types where InsP 3 and SOC are not active.

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca 2+ entry in thyroid FRTL-5 cells

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca 2+ concentrations ([Ca 2+] i) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca 2+ (SOC) entry was investigated. C1CP evoked a modest increase in [Ca 2+] i. The increase was inhibited by the SOC channel antagonist 1-(β-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1 H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca 2+] i was enhanced in comparison to control cells. When Ca 2+ was added to cells treated with both C1CP and thapsigargin in a Ca 2+-free buffer, the increase in [Ca 2+] i was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential ( E m) of the cells and attenuated the thapsigargin-invoked depolarization of the E m. The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl − ions, as C1CP in a Cl −-free solution also enhanced Ca 2+ entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca 2+] i nor did it modulate the E m. In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E m and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca 2+ ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.

Essential control of an endothelial cell ISOC by the spectrin membrane skeleton.

Mechanism(s) underlying activation of store-operated Ca2+ entry currents, I SOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and I SOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell I SOC (−45 pA at −80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 μM). Disruption of the spectrin–protein 4.1 interaction at residues A207-V445 of βSpIIΣ1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell I SOC, without altering activation of a nonselective current through cyclic nucleotide–gated channels. In contrast, disruption of the spectrin–actin interaction at residues A47-K186 of βSpIIΣ1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit I SOC. Results indicate that the spectrin–protein 4.1 interaction selectively controls I SOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.

Functional and molecular evidence of SOC channels in osteoblasts of rats: modulation of 1α,25-dihydroxyvitamin D 3

Functional and molecular evidence of SOC channels in osteoblasts of rats: modulation of 1α,25-dihydroxyvitamin D 3

Permeation of Monovalent Cations through the Non-capacitative Arachidonate-regulated Ca2+ Channels in HEK293 Cells: COMPARISON WITH ENDOGENOUS STORE-OPERATED CHANNELS

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.

Multiple effects of caffeine on Ca2+release and influx in human B lymphocytes

Caffeine has been used as a pharmacological tool to study the ryanodine receptor (RYR)-mediated Ca2+release from caffeine-sensitive, inositol 1,4,5,-trisphosphate (IP3)-insensitive pools. In the present study, we demonstrate multiple effects of caffeine on Ca2+homeostasis in human B lymphocytes. Although B cells express a functional RYR, which can be activated by 4-chloro-m-cresol following depletion of IP3-sensitive pools, caffeine does not activate RYR-mediated Ca2+release. Instead, caffeine dose-dependently inhibited IP3receptor (IP3R)-mediated Ca2+release, RYR-mediated Ca2+release and B cell receptor-initiated Ca2+influx, while high concentrations of caffeine (≥ 25mM) induced a Ca2+influx. In contrast with its ability to suppress receptor-stimulated Ca2+influx, caffeine had no significant effect on the store-operated Ca2+(SOC) channel-dependent Ca2+influx induced by thapsigargin. Thus, caffeine may act as an inhibitor on a single or multiple site(s) responsible for regulating the IP3R channel, RYR channel and presumably the receptor-mediated SOC channel. The present report may be the first demonstration of multiple effects of caffeine on Ca2+mobilization in single cell type. Our results suggest the need for caution regarding use of caffeine simply as a RYR-activator to study Ca2+homeostasis in eucaryotic cells.

Negative control of store-operated Ca2+ influx by B cell receptor cross-linking.

An increase in the intracellular Ca(2+) concentration by B cell receptor (BCR) cross-linking plays important roles in the regulation of B cell functions. [Ca(2+)](i) is regulated by Ca(2+) release from the Ca(2+) store as well as store-operated Ca(2+) influx (SOC). Protein tyrosine kinases downstream of BCR cross-linking were shown to regulate the mechanism for Ca(2+) release. However, it remains elusive whether BCR cross-linking regulates SOC or not. In this study, we examined the effect of BCR cross-linking on thapsigargin-induced SOC in the DT40 B cells. We found that the SOC-mediated increase in intracellular Ca(2+) concentration was inhibited by BCR cross-linking. Using a membrane-potential-sensitive dye, we found that BCR cross-linking induced depolarization, which is expected to decrease the driving force of Ca(2+) influx and SOC channel conductance. When membrane potential was held constant by the transmembrane K(+) concentration gradient in the presence of valinomycin, the BCR-mediated inhibition of SOC was still observed. Thus, the BCR-mediated inhibition of SOC involves both depolarization-dependent and depolarization-independent mechanisms of SOC inhibition. The depolarization-independent inhibition of the SOC was abolished in Lyn-deficient, but not in Bruton's tyrosine kinase-, Syk- or SHIP (Src homology 2 domain containing phosphatidylinositol 5'-phosphatase)-deficient cells, indicating that Lyn is involved in the inhibition. These results show novel pathways of BCR-mediated SOC regulations.

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells

Sphingosine induces a biphasic increase in cytosolic-free Ca2+([Ca2+]i) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (β-aminoethyl ether) N, N, N′, N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca2+influx does not appear due to Ca2+influx through store-operated Ca2+(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca2+influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca2+]ielicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca2+from intracellular Ca2+stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca2+influx through an undefined Ca2+channel and cause a blockade of SOC channels.

1α,25-Dihydroxy-vitamin-D3-induced Store-operated Ca2+ Influx in Skeletal Muscle Cells: MODULATION BY PHOSPHOLIPASE C, PROTEIN KINASE C, AND TYROSINE KINASES

In skeletal muscle cells the steroid hormone 1alpha, 25-dihydroxy-vitamin-D3 (1,25(OH)2D3) nongenomically promotes Ca2+ release from intracellular stores and cation influx through both L-type and store-operated Ca2+ (SOC) channels. In the present work we evaluated the regulation and kinetics of the 1, 25(OH)2D3-stimulated SOC influx in chick muscle cells. Stimulation with 10(-9) M 1,25(OH)2D3 in Ca2+-free medium resulted in a rapid (40-60 s) but transient [Ca2+]i rise, which correlated with sterol-dependent inositol 1,4,5-trisphosphate production. The SOC influx stimulated by the hormone was insensitive to both L-type channel antagonists and polyphosphoinositide-specific phospholipase C (PPI-PLC) inhibitors but was fully inhibitable by La3+ and Ni2+. PPI-PLC blockade prior to 1,25(OH)2D3 stimulation suppressed both the [Ca2+]i transient and the SOC influx. 1,25(OH)2D3-induced SOC entry was markedly increased after 3 min of treatment (30% above basal) and then rapidly reached a steady-state level. The sterol-stimulated SOC influx was prevented by protein kinase C and tyrosine kinase inhibitors but unaffected by blockade of the protein kinase A pathway. None of these inhibitors altered the thapsigargin-induced SOC entry, suggesting the operation of a signaling mechanism different from that for sterol-dependent SOC influx. The present results indicate that 1,25(OH)2D3-induced activation of PPI-PLC is upstream to Ca2+ influx through SOC channels and point for a role of both protein kinase C and tyrosine kinases but not protein kinase A in the regulation of the sterol-dependent SOCE pathway.