Slow Calcium-dependent Inactivation of Depletion-activated Calcium Current. STORE-DEPENDENT AND -INDEPENDENT MECHANISMS
1995; Elsevier BV; Volume: 270; Issue: 24 Linguagem: Inglês
10.1074/jbc.270.24.14445
ISSN1083-351X
AutoresAdam Zweifach, Richard S. Lewis,
Tópico(s)Ion Channels and Receptors
ResumoFeedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of ~30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EGTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by ~50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals. Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of ~30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EGTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by ~50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals. INTRODUCTIONIn many nonexcitable cells, the depletion of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate (IP3)1 1The abbreviations used are: IP3inositol 1,4,5-trisphosphate[Ca2+]free intracellular Ca2+ concentrationICRACCa2+ release-activated Ca2+ currentTGthapsigarginCsAcyclosporin A. is the primary mechanism by which cell surface receptors activate Ca2+ influx across the plasma membrane(1Putney Jr., J.W. Bird G.S.J. Endocrinol. Rev. 1993; 14: 610-631Crossref PubMed Scopus (483) Google Scholar). This phenomenon was first proposed by Putney and termed capacitative Ca2+ entry(2Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1257) Google Scholar). Multiple types of ion channels underlying capacitative Ca2+ entry have been identified in different cells on the basis of their Ca2+ permeability and activation by agents that empty Ca2+ stores (for review, see Ref. 3Fasolato C. Innocenti B. Pozzan T. Trends Pharmacol. Sci. 1994; 15: 77-83Abstract Full Text PDF PubMed Scopus (437) Google Scholar). The depletion-activated Ca2+ channels present in mast cells and T cells, referred to as calcium release-activated Ca2+ (CRAC) channels, are distinguished from other types of depletion-activated Ca2+ channels by their high selectivity for Ca2+ over monovalent and other divalent cations(4Lewis R.S. Cahalan M.D. Cell Regul. 1989; 1: 99-112Crossref PubMed Scopus (354) Google Scholar, 5Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1478) Google Scholar, 6McDonald T.V. Premack B.A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar, 7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (690) Google Scholar, 8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar, 9Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar) and their extremely small unitary conductance(7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (690) Google Scholar, 8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar). CRAC channels have been shown to underlie the mitogen-stimulated Ca2+ influx that is essential for T cell activation following T cell receptor engagement(7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (690) Google Scholar, 9Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar). Furthermore, periodic changes in CRAC channel activity have been shown to generate oscillations in the level of intracellular free Ca2+ ([Ca2+]i)(4Lewis R.S. Cahalan M.D. Cell Regul. 1989; 1: 99-112Crossref PubMed Scopus (354) Google Scholar, 10Dolmetsch R. Lewis R.S. J. Gen. Physiol. 1994; 103: 365-388Crossref PubMed Scopus (151) Google Scholar), which may serve to enhance signaling through the T cell receptor(11Negulescu P.A. Shastri N. Cahalan M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2873-2877Crossref PubMed Scopus (227) Google Scholar).The signal that couples store depletion to the activation of CRAC channels has not yet been identified, and in principle it could encompass diffusible and/or membrane-associated molecules. In a recent study, Randriamampita and Tsien (12Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (785) Google Scholar) isolated a fraction from the cytoplasm of stimulated Jurkat leukemic human T cells that triggers Ca2+ influx when applied to the exterior of astrocyte, neuroblastoma, and macrophage cell lines, apparently without releasing Ca2+ from stores. This activity was attributed to calcium influx factor, a small (<500 Mr) nonproteinaceous phosphate-containing factor that they proposed as a diffusible messenger responsible for activating capacitative Ca2+ entry. Additional activation mechanisms involving GTP-binding proteins(13Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 14Bird G.S.J. Putney Jr., J.W. J. Biol. Chem. 1993; 268: 21486-21488Abstract Full Text PDF PubMed Google Scholar, 15Jaconi M.E.E. Lew D.P. Monod A. Krause K.-H. J. Biol. Chem. 1993; 268: 26075-26078Abstract Full Text PDF PubMed Google Scholar), tyrosine kinases(16Sargeant P. Farndale R.W. Sage S.O. J. Biol. Chem. 1993; 268: 18151-18156Abstract Full Text PDF PubMed Google Scholar), cGMP(17Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 18Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar), and direct physical coupling between the store's membrane and the plasma membrane (19Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar) have been proposed (for reviews, see Refs. 1Putney Jr., J.W. Bird G.S.J. Endocrinol. Rev. 1993; 14: 610-631Crossref PubMed Scopus (483) Google Scholar and 3Fasolato C. Innocenti B. Pozzan T. Trends Pharmacol. Sci. 1994; 15: 77-83Abstract Full Text PDF PubMed Scopus (437) Google Scholar).Little is known about how CRAC channels are turned off once activated, although several reports have demonstrated that increased [Ca2+]i may play a role. Feedback inhibition by intracellular Ca2+ appears to occur through multiple mechanisms. After entering the cell, Ca2+ binds to sites probably residing on the CRAC channel itself(20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar), eliciting rapid inactivation over tens of milliseconds(8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar, 20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar). In addition, slow inactivation of ICRAC on a time scale of seconds has been observed following abrupt elevation of [Ca2+]o or global rises in [Ca2+]i(4Lewis R.S. Cahalan M.D. Cell Regul. 1989; 1: 99-112Crossref PubMed Scopus (354) Google Scholar, 6McDonald T.V. Premack B.A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar, 7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (690) Google Scholar, 8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar), but the underlying mechanisms have not been determined. A central tenet of the capacitative Ca2+ entry hypothesis is that refilling of intracellular Ca2+ stores should terminate Ca2+ influx. This prediction has been confirmed in intact cells using fluorescent Ca2+ indicators(10Dolmetsch R. Lewis R.S. J. Gen. Physiol. 1994; 103: 365-388Crossref PubMed Scopus (151) Google Scholar, 21Jacob R. J. Physiol. (Lond.). 1990; 421: 55-77Crossref Scopus (203) Google Scholar, 22Montero M. Alvarez J. Garca-Sancho J. Biochem. J. 1992; 288: 519-525Crossref PubMed Scopus (42) Google Scholar). However, the ability of store refilling to close CRAC channels, which is an important test of the role of CRAC channels in capacitative Ca2+ entry, has not yet been examined directly in patch-clamp experiments. Furthermore, the presence of additional negative feedback pathways through which Ca2+ may turn off CRAC channels has not been explored.This paper describes two mechanisms by which elevated [Ca2+]i slowly feeds back to inactivate CRAC channels. One mechanism is dependent on store refilling, while the other operates even when refilling is completely prevented with thapsigargin. Thus, with the inclusion of fast inactivation(8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar, 20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar), it appears that intracellular Ca2+ feeds back by at least three independent pathways to control capacitative Ca2+ entry. Autoregulatory feedback on CRAC channels by Ca2+ is likely to be essential in determining the character of receptor-stimulated Ca2+ signals in nonexcitable cells. A portion of this work has been reported previously in abstract form (23Zweifach A. Lewis R.S. Biophys. J. 1994; 66: A153Abstract Full Text PDF PubMed Scopus (36) Google Scholar).EXPERIMENTAL PROCEDURESCells and ReagentsJurkat E6-1 human leukemic T cells were maintained in complete medium containing RPMI 1640 and 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 25 mM HEPES, in a 6% CO2 humidified atmosphere at 37°C. Log phase cells (0.2-1.2 × 106/ml) were used in all experiments. Thapsigargin, okadaic acid, 1-norokadaone, and calyculin A (LC Pharmaceuticals, Woburn, MA) were prepared as stocks of 1 mM or 100 μM in Me2SO. Cyclosporin A (2 mg/ml in 2% ethanol) and FK506 (50 μg/ml in ethanol) were the generous gift of Drs. N. Clipstone and G. Crabtree at Stanford University.Patch-Clamp RecordingPatch-clamp experiments were conducted in the standard whole cell recording configuration(24Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15093) Google Scholar). Extracellular Ringer's solution contained the following: 155 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 or 22 mM CaCl2, 10 mMD-glucose, and 5 mM Na-HEPES (pH 7.4). Ca2+-free Ringer's contained 3 mM MgCl2. Internal solutions contained the following: 140 mM cesium aspartate, 10 mM Cs-HEPES (pH 7.2), and either 0.66 mM CaCl2, 11.68 mM EGTA, and 3.01 mM MgCl2 (12 mM EGTA solution) or 0.066 mM CaCl2, 1.2 mM EGTA, and 2.01 mM MgCl2 (1.2 mM EGTA solution). Free [Ca2+] in both of these solutions as measured with indo-1 was 5 nM; free [Mg2+] was calculated to be 2 mM. Recording electrodes were pulled from 100-μl pipettes (VWR), coated with Sylgard near their tips, and fire-polished to a resistance of 2-6 megaohms when filled with cesium aspartate pipette solution. The patch-clamp output (Axopatch 200, Axon Instruments, Foster City, CA) was filtered at 1.5 kHz with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at a rate of 5 kHz. Stimulation and recording were performed with an Apple Macintosh computer driving an ITC-16 interface (Instrutech, Elmont, NY) and using PulseControl software extensions (Jack Herrington and Richard Bookman, University of Miami) to Igor Pro (WaveMetrics, Inc., Lake Oswego, OR). Command potentials were corrected for the −12 mV junction potential that exists between the aspartate-based pipette solutions and Ringer's solution. Cells were allowed to settle onto but not firmly adhere to glass coverslip chambers shortly prior to each experiment. Adherent and nonadherent cells behaved identically in experiments with 1.2 mM EGTAi; however, currents obtained with 12 mM EGTAi were not as sustained in adherent cells as in nonadherent ones. The reason for this difference was not investigated further. ICRAC was induced through the store depletion protocol described previously(20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar). Following formation of the gigaseal, each cell was exposed to Ca2+-free Ringer's, and the whole cell recording configuration was established. This procedure was sufficient to activate ICRAC maximally, because additional pretreatment with 1 μM thapsigargin did not further increase the average initial size of Ca2+ current (Table I). After 3 min, [Ca2+]owas elevated to 2-22 mM, and steady-state ICRAC was measured in all experiments as a 5-10-ms average at the end of 200-ms pulses from −12 mV (the holding potential) to −132 mV delivered once every 2 s. In the experiment shown in Fig. 1, peak ICRAC was measured from a 1-ms average beginning 3 ms after the start of each hyperpolarizing pulse to minimize contributions from uncompensated capacitative current (time constant, <1 ms) and fast inactivation. All data are corrected for leak and residual capacitative current measured in the absence of Cao2+. Leak conductances were 20-100 picosiemens. Series resistance compensation was not employed, since the series resistance (4-25 megaohms) produced voltage errors of <3 mV. Cell capacitance was determined either from the settings of the whole cell capacitance-compensation circuitry, or by integrating currents elicited by 10-mV depolarizing steps. External solutions were changed by positioning the cell ~1 mm inside one barrel of a perfusion tube array through which the desired solutions flowed (<0.1 ml/min). Experiments were conducted at 22-25°C. Where normalized data are presented, current amplitudes were divided by the current's maximal value, and the time at which the maximum occurred was defined as time zero. Data are presented as mean ± S.E. Statistical significance of results was assessed using the Mann-Whitney U test, and differences are considered significant if p < 0.01. [Ca2+]i Measurements with Indo-1-For experiments combining patch-clamp recording with [Ca2+]i measurements, pipette solutions were supplemented with 100 μM indo-1 pentapotassium salt (Molecular Probes, Eugene, OR). Recording conditions have been described in detail previously(20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar). Briefly, cells were illuminated using a 75W xenon arc lamp and a 360 ± 5-nm interference filter (Omega Optical, Brattleboro, VT) mounted on a Nikon Diaphot inverted microscope equipped with a Nikon Fluor 40× objective (numerical aperature, 1.3) and a transistor-transistor logic-controlled shutter to control the duration of illumination. The emission signal was collected from an area adjusted to be slightly larger than the cell. Emitted light was split with a 440-nm dichroic mirror and passed through 405 ± 15 and 480 ± 12.5 nm interference filters (Chroma Technology Corp., Brattleboro, VT) to two photomultiplier tubes (HC124-02, Hamamatsu Corp., Bridgewater, NJ). [Ca2+]i was estimated from the relation [Ca2+]i = K∗(R − Rmin)/(Rmax - R). Background fluorescence was measured in the cell-attached mode and was subtracted from subsequent fluorescence signals before calculation of the 405/480 ratio, R. K∗, Rmin, and Rmax were determined from in vivo calibrations as described previously(20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar).Table IICRAC and [Ca2+]i following store depletionFigure 1:Fast and slow inactivation of ICRAC. A, after depletion of intracellular Ca2+ stores, exposure of a Jurkat T cell to 22 mM Cao2+ induces a slowly decaying inward current. Data points were measured every 2 s from the peak current (○) or steady-state current (□) elicited during brief hyperpolarizing voltage pulses shown in B (see "Experimental Procedures"). Collection times for the traces in B and C are indicated by i-iii and a-c, respectively. B, selected current responses to 200-ms voltage pulses from −12 to −132 mV. Rapid inactivation of ICRAC is induced by the sudden increase in Ca2+ influx upon hyperpolarization. Dashed line indicates zero current level. C, current/voltage relation for the inward current. Responses to voltage ramps (200-ms duration) from −120 to +50 mV show that current is inward in this voltage range as expected for ICRAC. Data in B and C are not corrected for leak or capacitative currents. Internal solution, cesium aspartate + 1.2 mM EGTA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSFast and Slow Inactivation of ICRAC by Intracellular Ca2+ICRAC was induced in Jurkat leukemic T cells by the passive depletion of intracellular Ca2+ stores. Depletion was achieved in these experiments by incubating each cell in Ca2+-free Ringer's solution for 3 min while dialyzing its interior with a pipette solution in which [Ca2+] was buffered to 5 nM. After such treatment, elevation of [Ca2+]o from 0 to 22 mM rapidly elicited an inward current (Fig. 1) whose properties identified it as ICRAC(4Lewis R.S. Cahalan M.D. Cell Regul. 1989; 1: 99-112Crossref PubMed Scopus (354) Google Scholar, 5Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1478) Google Scholar, 6McDonald T.V. Premack B.A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar, 7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (690) Google Scholar, 8Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (656) Google Scholar, 9Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar, 20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar). These properties included a dependence on Cao2+ (Fig. 1A), rapid inactivation during hyperpolarizing voltage pulses (Fig. 1B), an inwardly rectifying current-voltage relation with no clear reversal potential up to +50 mV (Fig. 1C), voltage-independent gating, and a lack of significant current noise. As illustrated in Fig. 1A, ICRAC decayed slowly in cells dialyzed with a relatively low amount of EGTA (1.2 mM). In this paper, we refer to the slow decay in current as slow inactivation; the use of this terminology is not intended to imply any specific type of mechanism. In all experiments, we measured ICRAC during brief hyperpolarizing voltage pulses to −132 mV delivered every 2 s (as in Fig. 1B). This protocol optimizes the size of the current and allows independent measurement of fast and slow inactivation, as described previously(20Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (330) Google Scholar). The decay in the peak currents (Fig. 1A, squares) reflects the slow inactivation process alone, whereas decay of the steady-state currents (Fig. 1A, circles) is determined by both fast and slow inactivation. Thus, the fact that both measures of ICRAC decay with the same time course suggests that the fast and slow inactivation processes are independent. In further support of this conclusion, none of the pharmacological treatments described below affected fast inactivation.[Ca2+]i and current measurements were combined in indo-1-loaded cells to examine the calcium dependence of slow inactivation. When depleted cells dialyzed with low [EGTA]i(1.2 mM) were exposed to 22 mM Cao2+, [Ca2+]i remained relatively constant for ~10 s before climbing to micromolar levels, as would be expected since Ca2+ influx via ICRAC should eventually overcome the capacity of EGTA to buffer Ca2+ (Fig. 2A). The increase in [Ca2+]i was associated with a progressive inactivation of ICRAC over a period of ~100 s. As the current declined, [Ca2+]i reached a peak and decreased, presumably as the rate of Ca2+ entry fell below that of Ca2+ efflux by Ca2+-ATPases in the plasma membrane. Increasing the buffering power of the pipette solution with 12 mM EGTA restricted [Ca2+]i to levels below 100 nM for the duration of the experiment and reduced the slow decay of the current (Fig. 2B). The ability of increased intracellular Ca2+ buffering to prolong the current is summarized for 10-17 experiments in Fig. 2C. These results demonstrate that slow inactivation is Cai2+-dependent.Figure 2:Slow inactivation of ICRAC is Ca2+-dependent. A, in the presence of 1.2 mM EGTA, the induction and subsequent slow decline of ICRAC is associated with a delayed rise and fall of [Ca2+]. B, 12 mM EGTA in the pipette largely suppresses the [Ca2+] increase, and ICRAC is more sustained. All currents shown in this and subsequent figures were measured at the end of hyperpolarizing voltage pulses to −132 mV as described in Fig. 1B. C, the effect of intracellular buffering on slow inhibition of ICRAC from experiments like those in A and B. Current amplitude is normalized to the maximum amplitude reached in each experiment after exposure to 22 mM Cao2+, and time is plotted from this point onward. Plotted values are the mean ± S.E. of 10-17 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)During whole cell recording, soluble components diffuse out of the cell and may cause rundown of channel activity. To test whether slow inactivation of ICRAC is due to a washout phenomenon, we examined whether it can be reversed by removal of extracellular Ca2+. Inhibition of ICRAC in the presence of 22 mM Cao2+ was allowed to reach steady state; at this point, Cao2+ was removed for a variable period and then reapplied to measure the extent to which ICRAC had recovered. In the cell depicted in Fig. 3, a 60-s exposure to Ca2+-free conditions allowed nearly complete recovery of the current's amplitude. The extent of recovery varied among the cells tested. In six cells in which slow inactivation reduced ICRAC to a level of 11 ± 3%, subsequent incubation in 0 Cao2+ for 100-150 s allowed recovery to 54 ± 14% of the initial peak amplitude. The source of the variation is not known. However, evidence presented below suggests that most of the inactivation in these experiments is due to reuptake of Ca2+ by stores; thus, a variable degree of reemptying following removal of Cao2+ may contribute to the different amounts of recovery that were observed. Regardless, these results further support the Ca2+ dependence of slow inactivation and demonstrate that it is not simply due to nonspecific rundown or to washout of a factor essential for maintenance of ICRAC.Figure 3:Slow inactivation is reversible. After ICRAC declined to a steady-state level in 22 mM Cao2+, the cell was bathed in Ca2+-free Ringer's solution for 60 s and then reexposed to 22 mM Ca2+. In this cell, the current recovered almost completely, indicating that inactivation is not due to washout of a diffusible factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Refilling of Ca2+Stores Contributes to Slow InactivationIn the experiments described above, Ca2+ stores were depleted passively, without increasing the permeability of the endoplasmic reticulum membrane to Ca2+ or inhibiting its Ca2+-ATPases. Under these conditions, stores would be expected to refill efficiently following a rise in [Ca2+]i, thereby causing the slow inactivation of ICRAC. To test this hypothesis, we measured the time course of ICRAC in the presence of a high dose of TG (1 μM) that fully blocks Ca2+-ATPases in the endoplasmic reticulum (9Premack B.A. McDonald T.V. Gardner P. J. Immunol. 1994; 152: 5226-5240PubMed Google Scholar, 10Dolmetsch R. Lewis R.S. J. Gen. Physiol. 1994; 103: 365-388Crossref PubMed Scopus (151) Google Scholar, 25Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar) and hence prevents Ca2+ reuptake. Cells were incubated in Ca2+-free Ringer's + TG for 3 min prior to exposure to 22 mM Cao2+. Under these conditions with 1.2 mM EGTAi, slow inactivation of ICRAC was greatly reduced despite a large increase in [Ca2+]i (Fig. 4A, Table I). In 21 cells treated with TG, ICRAC decayed over 100 s to a steady-state level of ~50% of its initial value (Fig. 4B). Consistent with the time course of the current, [Ca2+]i declined only partially over the same time period (Fig. 4A, Table I). These results demonstrate that store refilling contributes to slow Ca2+-dependent inactivation of ICRAC. The failure of TG to fully hinder this process is not due to an inability to prevent store refilling. Experiments with TG were repeated with 20 μM IP3 in the internal solution to attempt to increase the overall extent of store depletion. The release of stored Ca2+ by 20 μM IP3 was confirmed in separate experiments by a large [Ca2+]i spike occurring within seconds of breaking into cells in Ca2+-free Ringer's; such transients were not observed in the absence of IP3 (data not shown). Furthermore, IP3 alone prevented slow inactivation to about the same extent as TG alone (~50%; n = 3 cells). As summarized in Fig. 4B, IP3 and TG together had the same effect as TG alone, indicating that store refilling is not occurring and therefore cannot explain the failure of TG to prevent ICRAC inactivation. Rather, these results reveal a second process of Ca2+-dependent slow inactivation occurring independently of changes in Ca2+ store content.Figure 4:Store refilling contributes to slow inactivation. A, 1 μM TG partially blocks the slow decline in ICRAC and [Ca2+] in one cell exposed to 22 mM Cao2+ with 1.2 mM EGTA. TG was present during the 3-min preincubation in Ca2+-free Ringer's. B, TG limits slow inactivation to an average final extent of ~50%. Addition of 20 μM IP3 to the internal solution produces no further effect, indicating that incomplete emptying of stores is not responsible for the partial effect of TG on current inhibition. Data are the average normalized currents ± S.E. from 14-21 cells in experiments like those shown in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Phosphatase Inhibitors on Slow InactivationOkadaic acid, a potent inhibitor of phosphatases 1 and 2A (26Nishiwaki S. Fujiki H. Suganuma M. Furuya-Suguri H. Matsushima R. Iida Y. Ojika M. Yamada K. Uemura D. Yasumoto T. Schmitz F.J. Sugimura T. Carcinogenesis. 1990; 11: 1837-1841Crossref PubMed Scopus (125) Google Scholar, 27Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Abstract Full Text PDF PubMed Scopus (1256) Google Scholar) has been reported to enhance capacitative Ca2+ influx in Xenopus oocytes(28Parekh A.B. Terlau H. Stühmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar). We therefore tested the ability of okadaic acid and other phosphatase inhibitors to suppress the TG-insensitive component of slow inactivation. Okadaic acid inhibits protein phosphatases 1 and 2A in vitro with IC50 values of 1-50 nM(27Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Abstract Full Text PDF PubMed Scopus (1256) Google Scholar, 29Honkanen R.E. Codispoti B.A. Tse K. Boynton A.L. Toxicon. 1994; 32: 339-35
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