Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

ATP from subplasmalemmal mitochondria controls Ca2+-dependent inactivation of CRAC channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.     

Two types of store-operated channels in A431 cells

Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.     

Pathways of cadmium influx in mammalian neurons

The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2(+)-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 microM Cd2(+). This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.     

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.     

Close functional coupling between Ca2+ release-activated Ca2+ channels, arachidonic acid release, and leukotriene C4 secretion

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.     

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.     

Anion channels modulate store-operated calcium influx in human microglia

Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for entry of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effects on store-operated Ca2+ influx induced by replacement of NaCl with Na-gluconate in extracellular solutions. Three separate procedures were used to activate store-operated channels. Platelet activating factor (PAF) was used to generate a sustained influx of Ca2+ in standard physiological saline solution (PSS). The magnitude of this response was depressed by 70% after replacement of PSS with low Cl- PSS. A second procedure used ATP, initially applied in Ca2+-free PSS solution to deplete intracellular stores. The subsequent perfusion of PSS solution containing Ca2+ resulted in a large and sustained entry of Ca2+, which was inhibited by 75% with low Cl- PSS. The SERCA inhibitor cyclopiazonic acid (CPA) was used to directly deplete stores in zero-Ca2+ PSS. Following the introduction of PSS containing Ca2+, a maintained stores-operated influx of Ca2+ was evident which was inhibited by 77% in the presence of the low Cl- PSS. Ca2+ influx was linearly reduced with cell depolarization in elevated K+ (7.5 to 35 mM) suggesting that changes in external Cl- were manifest as altered electrical driving force for Ca2+ entry. However, 50 mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolarization. Patch clamp studies showed low Cl-PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated with voltage ramps. The results demonstrate that Cl- channels play an important role in regulating store-operated entry of Ca2+ in human microglia.     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.     

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.     

Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool

The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.     

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.     

Rapid modulation of Ca2+ uptake in human jejunal enterocytes

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.     

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.