Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs

In cultured pituatary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca²⁺]_i) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca²⁺]_i and LH secretion. The spike phase of the [Ca²⁺]_i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca²⁺. In contrast, the prolonged phase of the Ca²⁺ signal depends exclusively on Ca²⁺ entry from the extracellular pool. The influx of Ca²⁺ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca²⁺]_i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca²⁺ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca²⁺ signaling and LH exocytosis. In contrast to [Ca²⁺]_i measurements in cell suspension, single cell Ca²⁺ measurements revealed the existence of a more complicated pattern of Ca²⁺ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca²⁺ spiking. In addition, analysis of the magnitudes of the magnitudes of the [Ca²⁺]_i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca²⁺, and to K⁺ and phorbol ester stimulation, showed non-linearity between these parameters with amplification of [Ca²⁺]_i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplication, especially during the plateau phase of the secretory response to GnRH.     

Effects of Basal [Ca]i on Calcium Handling in Ca-Overloaded Rat Cultured Heart Muscle Cells

To elucidate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺-signalling by the sarcoplasmic reticulum (SR) in Ca²⁺-overloaded heart muscle cells, the direct effects of “basal” [Ca²⁺]_i on calcium waves were investigated by altering the membrane potential. When basal inter-calcium wave (BCW) [Ca²⁺]_i was maintained at a high level, (i) calcium waves showed more gradual and more rapidly suppressed increase in [Ca²⁺]-profile (P < 0.005), and (ii) calcium waves occurred at a significantly higher frequency and velocity (259% and 137%), than when low BCW [Ca²⁺]_i was maintained. Similar investigations on inhibition of the Na⁺-Ca²⁺ exchanger, however, showed that membrane potential did not elicit direct effects on calcium waves. These results showed that the elevation of BCW [Ca²⁺]_i per se directly influences Ca²⁺-signalling in heart muscle cells through non-equilibrated release-restoration Ca²⁺-handling by the SR.     

Cytosolic Ca2+ as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin filaments and microtu-bules are usually employed to provide the structural ba-sis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the abil-ity to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly under-stood. Here we show that Ca²⁺ oscillations induced by the Ca²⁺ release from intracellular Ca²⁺ store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suumsperm activation. The chelation of cytosolic Ca²⁺ suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing ex-ogenous Ca²⁺ into sperm cells. Ca²⁺ promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca²⁺ promotes MSP disassembly by activating Ca²⁺/calmodulin-depend-ent serine/threonine protein phosphatase calcineurin. In addition, Ca²⁺/camodulin activity is required for the fusion of sperm-specific membranous organelle with the plasma membrane, a regulated exocytosis required for sperm mo-tility. Thus, Ca²⁺ plays multifunctional roles during sperm activation in Ascaris suum.     

Lysophosphatidic Acid Sensitises Ca Influx through Mechanosensitive Ion Channels in Cultured Lens Epithelial Cells

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca²⁺]_i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca²⁺]_i. The mechanical stress-induced increase in [Ca²⁺]_i in the presence of LPA was inhibited by removing extracellular Ca²⁺ or by addition of Gd³⁺, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca²⁺ influx through cation-selective mechanosensitive channels, but does not sensitise Ca²⁺ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca²⁺ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca²⁺ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca²⁺]_i induced by mechanical stress.     

Oxidative stress and Mrp2 internalization

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca²⁺ chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca²⁺-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 μM). Moreover, an increase in the intracellular Ca²⁺ level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca²⁺ elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.     

Modelling of calcium handling in airway myocytes

Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca²⁺]_i increase via Ca²⁺ release from the sarcoplasmic reticulum through InsP₃ receptors. Several models have been developed to explain the characteristics of InsP₃-induced [Ca²⁺]_i responses, in particular Ca²⁺ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca²⁺ handling, i.e., InsP₃ receptors, SERCAs, mitochondria and Ca²⁺-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca²⁺ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP₃ receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca²⁺ clearance in the pattern of [Ca²⁺]_i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca²⁺ influx. Stimulation of this model by simulated increase in [Ca²⁺]_i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca²⁺ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.     

Endothelin stimulates tyrosine phosphorylation of p125 and p130 in rat cerebral cortex

Stimulation of rat cerebral cortex with endothelin-1 (ET-1) caused an increase in the tyrosine phosphorylation of several proteins. Two of these phosphoproteins were identified by the immunoprecipitation assays as being the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. This effect was time- and dose-dependent, with an EC₅₀ value of 3.9×10⁻⁸ M. In addition, the cerebral cortex ET receptor subtype involved in this action was determined by using BQ-123 and BQ-788, which are ET_A and ET_B receptor antagonists respectively. Our results indicate that the ET-1 effect on protein tyrosine phosphorylation occurred through ET_B receptors. The requirement for extracellular Ca²⁺ on ET-1 action was also studied. ET-1-stimulated tyrosine phosphorylation of both p125^FAK and p130^Cas was abolished in the absence of external Ca²⁺ or in the presence of nimodipine, a Ca²⁺ channel-blocker. These results suggest that the ET-1-stimulated protein tyrosine phosphorylation was secondary to Ca²⁺ influx through the dihydropyridine Ca²⁺-channel. In slices where protein kinase C was inhibited, ET-1-stimulated tyrosine phosphorylation of both proteins was reduced. These results indicate that ET-1 modulates the tyrosine phosphorylation of specific proteins, which may be involved in adhesion processes in the brain.     

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death

Mitochondrial calcium uniporter (MCU) is a conserved Ca²⁺ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stressinduced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca²⁺ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca²⁺ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.     

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: Regulation by calcium,protein kinases,and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actincrosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5–10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin. © 1996 Wiley-Liss, Inc.     

Neurotensin elevates cytosolic calcium in small cell lung cancer cells

The ability of neurotensin (NT) to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca²⁺ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca²⁺ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8–13) but not N-terminal fragments such as NT(1–8) elevated the cytosolic Ca²⁺ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca²⁺ from intracellular stores. These data indicate that SCLC NT receptors may use Ca²⁺ as a second messenger.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Tumour-promoting phorbol esters inhibit agonist-induced phosphatidate formation and Ca flux in human platelets

Tumour-promoting phorbol esters (phorbol-12-myristate-13-acetate, PMA; phorbol-12,13-dibutyrate, PDBu) but not 4β-phorbol, activate protein kinase C. Using human platelets pre-labelled with quin2 or ³²PO₄ we examined the effects of these compounds on human platelet cytosolic free Ca²⁺ ([Ca²⁺]_j) and on [³²]phosphatidic acid ([³²P]PtdOH). PMA and PDBu, but not 4β-phorbol inhibited thrombin-, PAF- and vasopressin-induced elevation of [Ca²⁺], and [²⁺P]PtdOH formation. It is suggested that protein kinase C may act to terminate the transduction processes that link receptor occupancy to cellular activation.     

Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: Role of calcium

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [¹⁴C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E₂ biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A₂ activity is Ca²⁺-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca²⁺ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca²⁺ concentration; when external Ca²⁺ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca²⁺ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca²⁺ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca²⁺ ([Ca²⁺])_i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca²⁺]_i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca²⁺ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca²⁺ level.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Vasoactive intestinal polypeptide stimulates cyclic AMP production in mouse N1E-115 neuroblastoma cells: Modulation by a protein kinase C activator and ionomycin

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca²⁺ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4β-phorbol 12-myristate 13-acetate reduced VIP-but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca²⁺ potentiates this signaling pathway.     

Two bombesin analogues discriminate between neuromedin B- and bombesin-induced calcium flux in a lung cancer cell line

We examined the profile of two bombesin (BN) antagonists, (CH₃)₂CHCO-His-Trp-Ala-Val- -Ala-His-Leu-NHCH₃] (ICI 216140) and [ -Phe⁶,des-Met¹⁴]BN(6–14)ethylamide (DPDM-BN EA), against neuromedin B-induced Ca²⁺ mobilization in the small cell lung cancer (SCLC) line NCI-H345. Neuromedin B (NMB), a BN-like peptide sharing sequence homology with ranatensin, elicited a concentration-dependent Ca²⁺ release (in part) from intracellular stores. Sequential addition of NMB attenuated Ca²⁺ mobilization. Desensitization occurred between BN and NMB; depletion of intracellular Ca²⁺ is a likely mechanism because thapsigargin stimulated Ca²⁺ release after a maximally desensitizing dose of NMB. ICI 216140 and DPDM-BN EA competitively inhibited BN-induced Ca²⁺ transients. In contrast, these compounds antagonized NMB-stimulated Ca²⁺ transients in a noncompetitive manner. The pharmacological profiles obtained support receptor heterogeneity for BN-like peptides on this SCLC line, underscoring the need for thorough examination of dose-response relationships when investigating effects of BN analogues on intact cells.     

植物感受盐胁迫及相关钙信号的研究进展

盐胁迫对植物的生长和发育造成严重影响,其危害包括渗透胁迫、离子毒害等,严重损害了农业生产和粮食安全。在盐胁迫下,植物相关感受器接受刺激,使得Ca²⁺通过细胞膜以及细胞内钙库膜上打开的Ca²⁺通道进入细胞质基质,导致细胞质内Ca²⁺浓度升高,产生钙信号。钙离子作为重要的第二信使,在植物细胞内和细胞间传递信号,信号往下游传递,在不同生长和发育阶段引起植物一系列的生理响应来应对盐胁迫影响。钙信号主要通过钙调蛋白（CaM）、钙调素样蛋白（CML）、钙依赖性蛋白激酶（CDPK）、钙调磷酸酶B样蛋白（CBL）和CBL互作蛋白激酶（CIPK）感知并将特异的钙信号信息传递到下游;从而激活植物盐胁迫生理响应。本文主要综述植物如何感知盐胁迫刺激,以及钙信号产生与传导机制,并对该研究领域需解决的问题进行了展望。     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Depletion of intracellular calcium stores activates an outward potassium current in mast and RBL-1 cells that is correlated with CRAC channel activation

Highly Ca²⁺ selective Ca²⁺ channels activated by store depletion have been recently described in several cell types and have been termed CRAC channels (for calcium release-activated calcium). The present study shows that following store depletion in mast and RBL-1 cells, monovalent outward currents could be recorded if the internal solution contained K⁺ but not Cs⁺. The activation of the outward K⁺ current correlated with the activation of I_CRAC, in both time and amplitude, suggesting that the K⁺ current might be carried by CRAC channels. The amplitude of the outward current was increased if external Ca²⁺ was reduced or replaced by external Ba²⁺. The outward K⁺ conductance might have a physiological role in maintaining the driving force for Ca²⁺ entry during the activation of CRAC channels.