Stable Activation of Single Ca2+ Release-activated Ca2+ Channels in Divalent Cation-free Solutions
2001; Elsevier BV; Volume: 276; Issue: 2 Linguagem: Inglês
10.1074/jbc.m008348200
ISSN1083-351X
AutoresFranz-Josef Braun, Lisa M. Broad, David L. Armstrong, James W. Putney,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoThe regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca2+, Mg2+, and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca2+stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca2+, and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg2+ present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836–839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion. The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca2+, Mg2+, and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca2+stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca2+, and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg2+ present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836–839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion. inositol 1,4,5-trisphosphate store-operated channel rat basophilic leukemia cells 2-aminoethoxydiphenyl borane 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid Ca2+ release-activated Ca2+channel In a variety of cells, binding of ligands such as neurotransmitters, hormones, or growth factors to receptors on the cell surface generates intracellular calcium signals. Receptor-mediated release of Ca2+ from IP31-sensitive intracellular stores triggers an influx of Ca2+ into the cell via store-operated channels (SOCs) in the plasma membrane, a process termed "capacitative calcium entry" (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2109) Google Scholar, 2Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). The resulting increase in intracellular Ca2+ concentration regulates important processes, ranging from cell growth and differentiation to apoptosis and cell death. Molecular candidates for SOCs are the mammalian homologues of the Drosophila TRP (transient receptor potential) protein (3Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (356) Google Scholar). Expression of the related genes, however, has resulted in varying patterns of response, including constitutive activity, augmentation of capacitative calcium entry, apparent direct activation by IP3, and activation by diacylglycerol (4Putney Jr., J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar). Therefore, the mechanism by which emptying of intracellular Ca2+ stores activates SOCs remains elusive. Recent findings have favored a "direct coupling" model, whereby IP3 receptors in the store membranes sense the Ca2+-filling status of the stores and transfer this information via direct coupling to subunits of SOCs in the plasma membrane (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (579) Google Scholar, 6Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1049) Google Scholar, 7Zubov A.I. Kaznacheeva E.V. Alexeeno V.A. Kiselyov K. Muallem S. Mozhayeva G. J. Biol. Chem. 1999; 274: 25983-25985Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 8Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). For the present, the best knowledge of SOCs comes from electrophysiological studies of a specific current associated with SOCs, known as the Ca2+ release-activated calcium current (Icrac) (9Hoth M. Penner R. Nature. 1992; 355: 353-355Crossref PubMed Scopus (1491) Google Scholar). Despite extensive characterization of the macroscopic CRAC currents, the single-channel signature is poorly understood. This is because the channels are presumed to have a minute single-channel conductance, too low to be resolved at the single-channel level (10Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (695) Google Scholar). During whole cell measurements in Jurkat T-lymphocytes, recently, Kerschbaum and Cahalan (11Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar) were able to detect for the first time single-channel events which they attributed to CRAC channels. Their approach was to measure currents in the complete absence of intra- and extracellular divalent cations. They reasoned that under these conditions CRAC channels, like voltage-gated Ca2+ channels should pass monovalent cations indiscriminately and with enhanced single-channel conductance (12Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (661) Google Scholar, 13Lepple-Wienhues A. Cahalan M.D. Biophys. J. 1996; 71: 787-794Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 14Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 1998; 111: 521-537Crossref PubMed Scopus (85) Google Scholar). Indeed, in the absence of divalent cations large nonselective cation currents were observed, and during the initial stage of activation, single-channel conductances of 36 to 40 pS were measured in whole cell mode (11Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar). However, the relationship of these large nonselective cation currents to the process of store depletion, and thus their identity as CRAC channels, has not been definitively established. Here we report similar single-channel activities measured for the first time in excised plasma membrane patches, using a mast cell line (RBL-2H3 m1) and divalent cation-free solutions. Our whole cell data are similar in some respects to those from Jurkat T-lymphocytes. In addition, because of direct access to the cytoplasmic surface of the channels, we have elucidated kinetic and regulatory properties of the channels. In particular, we have sought to determine whether or not these cation channels truly represent CRAC channels. In divalent cation-free solutions excised channels of 25 to 39 pS conductance showed stable activation, independent of IP3 or IP3 receptors, and were blocked by 2-APB, a relatively specific inhibitor of SOC activation (15Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar, 16Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (533) Google Scholar). The effects of 2-APB, as well as the modulation of channel activity by Ca2+ provide evidence that the Na+-conducting channels are in fact single CRAC channels. Hence, this report offers new insight into the possible regulation mechanism of native SOCs and provides a novel approach to investigate these channels under low divalent conditions. Rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line expressing m1 muscarinic receptors, were obtained from Dr. M. Beaven, National Institutes of Health (17Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595PubMed Google Scholar). The cells were cultured in Earle's minimal essential medium with Earle's salts, 10% fetal bovine serum, 4 mm l-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin (37 °C, 5% CO2). For experiments, cells were plated onto glass coverslips and used 12–36 h there after. Patch clamp experiments were performed at 20–22 °C in the tight-seal whole cell, cell-attached and inside-out configurations (18Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15145) Google Scholar). Patch pipettes were pulled from borosilicate glass (Corning glass, 7052) and fire polished. Membrane currents, filtered at 1–2 kHz, were recorded using an Axopatch-200B amplifier (Axon Instruments, Burlingame, CA). Voltage clamp protocols were implemented and data acquisition performed with pCLAMP 7.0 Software (Axon Instruments). Solution changes were accomplished by bath perfusion. The time required for a complete change was around 2 s. All voltages were corrected for a liquid junction potential. For whole cell experiments with Ca2+ as charge carrier, unless stated otherwise, the patch pipette (2–5 megaohm) solutions had the following composition (in mm): 140 Cs+-aspartate, 2 MgCl2, 1 MgATP, 10 Cs+-BAPTA (with free calcium set to 100 nm, calculated using MaxChelator software, version 6.60), 10 HEPES (pH 7.2 with CsOH). The bath solutions contained (in mm) 140 NaCl, 4.7 KCl, 10 CsCl, 1,13 MgCl2, 10 glucose, 10 CaCl2, 10 HEPES (pH 7.2 with NaOH). Divalent free whole cell measurements, with Na+ as charge carrier, were done with pipettes containing (in mm) 128 Cs+-aspartate, 12 Cs+-BAPTA, 0.9 CaCl2 (free Ca2+ ∼5 nm), 10 HEPES (pH 7.2 with CsOH), and bath solutions containing (in mm) 150 Na+ methane sulfonate, 2 EDTA, 10 HEPES (pH 7.2 with NaOH). CRAC channels were opened by passive store-depletion with high BAPTA (12 mm) in the pipette solution, or by addition of ionomycin or thapsigargin to the bath. Cells were held at a potential of 0 mV. Every 1, 2, or 5 s either voltage ramps from −100 to +60 mV or voltage steps from 0 to −100 mV were delivered for 200 ms. Currents were sampled at 5 kHz during voltage ramps and 25 kHz during voltage steps. All whole cell data were corrected for leak currents. Leak current was collected in response to voltage ramps or steps immediately after establishing the whole cell configuration. During cell-attached and inside-out recordings data were collected from 10-s records at the given membrane potential, digitized at 5 or 10 kHz and filtered digitally for analysis and presentation. The pipette (5–10 megaohm) solutions contained (in mm) 150 NaCl, 2 EDTA, 10 HEPES (pH 7.2 with NaOH). In cell-attached experiments the bath solution contained (in mm) 145 KCl, 5 NaCl, 10 MgCl2, 10 HEPES (pH 7.2 with KOH) to nullify the cell's resting potential. 90 s before excision of the patch, the bath was perfused with intracellular solution, which contained (in mm) 145 K+-glutamate, 5 NaCl, 2 EDTA, 10 HEPES (pH 7.2 with KOH). Single-channel analysis was performed with the pCLAMP 6 software. po values were calculated for consecutive 1-s periods. Ca2+ release-activated Ca2+currents can be activated by a variety of procedures that share the common property of emptying intracellular IP3-sensitive stores. Stores can be depleted actively by exposure to receptor agonists that elevate IP3 levels, external application of Ca2+-ionophores like ionomycin, or the presence of IP3 in the patch pipette solution (19Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1291) Google Scholar). Passive methods for store depletion rely on a constitutive and ill-defined leak of Ca2+ from stores. If store refilling is prevented by inhibition of SERCA pumps or by high concentrations of cytoplasmic Ca2+ chelators, the stores gradually lose their Ca2+. In the whole cell measurements shown in Fig.1, we used the latter method and activated CRAC channels by breaking into the cell, while including high concentrations of the Ca2+-chelator, BAPTA (12 mm), in the pipette solution. With external Ca2+ present (Fig. 1 a), Ca2+ ions permeate through the open CRAC channels and the Ca2+-influx during progressive channel activation could be monitored as a developing small inward current, of 30 to 60 pA (2–3 pA/pF) (n = 5). Single-channel currents underlying the macroscopic Ca2+ currents were too small to be resolved, even at −120 mV membrane potential. With divalent free solutions and Na+ as the permeant ion, however, single-channel events have been observed during whole cell measurements in Jurkat T-lymphocytes (11Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar). We first sought to determine whether similar currents could be observed with rat basophilic leukemia (RBL-2H3 m1) cells. The current development in Fig. 1 billustrates activation of a large inward current under divalent cation-free conditions (n = 6). The latency for current activation after break in, and the development time to reach the peak macroscopic current, were characteristically 1.5 to 2 times longer for the current in divalent-free solutions, than for Icrac in Ca2+-containing solutions. In the absence of divalent cations, dialysis of the cell with the pipette solution typically activated macroscopic currents up to 2000 pA (∼130 pA/pF), or ∼40-fold greater than Icrac. The current-voltage relationship of the fully developed current showed a modest inward rectification. External addition of 10 μm Mg2+ inhibited the monovalent currents in a voltage-dependent manner (data not shown). During the initial activation of the large cation current (see Fig.1 b), we were able to detect single-channel openings in RBL-2H3 m1 cells (Fig. 1 c). We recorded single-channel inward currents of 3.3 to 4 pA, during voltage steps to −100 mV, giving conductances of 33 to 40 pS (n = 6, assuming a reversible potential of 0 mV, see below). Based on the whole cell current amplitudes and the unitary current of the single-channels, we calculated the number of channels per cell to be 260 to 500 channels (n = 6). This suggests an average surface density of ∼0.22 channels per μm2. To gain access to the cytoplasmic surface of the channels, and to limit the number of investigated channels, we attempted to record the activity of individual channels in excised plasma membrane patches. Sodium ions (150 mm) were used as the primary charge carriers. Starting from cell-attached patches with no channel activity, we excised the patches into divalent cation-free solutions, while clamping the cell's membrane potential to −73 mV. In 65 out of 95 inside-out patches this procedure induced a channel activity, with an average conductance of 31 pS (25 pS to 39 pS) (Fig.2, a and b). We conclude that the channel activity seen after excision corresponds to the channels underlying the macroscopic current activated by divalent cation-free solutions; the single-channel conductance found in excised patches was similar to the conductance recorded in the whole cell mode (Fig. 1 c), the channels appear to have similar lack of selectivity, and the channels have similar, high open probability under total divalent cation-free conditions. When patches were excised into divalent-free media, the channels apparently needed no additional factors to be activated, such as IP3 or exogenously added IP3 receptors (7Zubov A.I. Kaznacheeva E.V. Alexeeno V.A. Kiselyov K. Muallem S. Mozhayeva G. J. Biol. Chem. 1999; 274: 25983-25985Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Kiselyov K.I. Semyonova S.B. Mamin A.G. Mozhayeva G.N. Pflügers Arch. 1999; 437: 305-314Crossref PubMed Scopus (43) Google Scholar). However, if fragments of endoplasmic reticulum stores were still attached to the patches, the absence of ATP, Ca2+, and Mg2+ from the intracellular solutions could have induced store depletion, and thus caused or contributed to channel activation. Continuous channel activity began an average of 70 to 80 s after excision (Fig. 2 c) and was stable during recording time for at least 20 min, even after extensive washing. In 32% of the experiments the progressive opening of 6 and more channels in a patch could be observed, which may suggest clustering of CRAC channels in specific membrane domains. We examined the single-channel properties of channels in inside-out patches, in divalent cation-free solutions. Fig. 3 a shows recordings of 2 active channels in an excised inside-out patch, at different membrane potentials. The channels had a high open probability (0.94 ± 0.03 at −113 mV, n = 5). The duration of the closures from the open state decreased with hyperpolarization, from τ = 1.19 ± 0.13 (± S.E.) ms at −73 mV to τ = 0.50 ± 0.06 (± S.E.) ms at −113 mV (n = 7 cells). From traces such as those shown in Fig. 3 a, current amplitudes were measured manually or determined from all points amplitude histograms (Fig. 3 b), over a voltage range from −113 to +67 mV. The resulting current-voltage relationships (Fig.3 c) gave an average single-channel conductance of 31 pS (25 to 39 pS). Moreover, the IV-relationship was linear with a reversal potential close to 0 mV. As the major cation in the intracellular solution was K+, while that in the pipette was Na+, this is indicative of a channel that passes monovalent cations indiscriminately (12Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (661) Google Scholar). The IV-relationship is similar to that for the whole cell current, except the latter is slightly inwardly rectifying, due to a small effect of voltage on open probability (not shown). Another specific channel feature, the mean open time, was calculated to be 14.9 ± 1.2 (± S.E.) ms (n = 7) (Fig. 3 d). The results in Fig. 1 show that the activation of the large cation current by divalent cation-free solutions was considerably slower than the activation of Icrac, despite the expectation that passive store emptying by BAPTA should occur at a similar rate in the two conditions. This would indicate that the activation of cation channels by divalent cation-free solutions involves a mechanism other than, or in addition to store depletion. Alternatively, it is possible that the absence of divalent cations slows the mechanism for coupling store depletion to channel activation. To address these possibilities, we carried out experiments in which Ca2+-stores were emptied and CRAC channels thus activated prior to going whole cell or excision into divalent cation-free media. Before sealing on the cells, we applied either 1 μm of the SERCA inhibitor, thapsigargin plus 500 nm of the ionophore ionomycin, or 500 nm ionomycin alone. The incubation time with these drugs was minimally 5 min, by which time stores should be completely depleted and the currents should have fully developed (21Huang Y. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 19554-19559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Subsequently, we patched the cells, and established either the whole cell configuration with either Ca2+ or Na+ as charge carriers, or excised the patch (inside-out mode) at −73 mV V m into divalent-free solutions. In the whole cell experiments, the pipette solutions included high concentrations of BAPTA to passively deplete stores in the control cells. Prior depletion of Ca2+-stores resulted in preactivation of Icrac with about 2 pA/pF (7/7) when measured with Ca2+ as the charge carrier (Fig.4, a and b, inset). However, directly after switching to Na+ in divalent free solutions, the current only increased 4-fold (Fig. 4 b, inset), corresponding to about 10% of activation compared with the peak macroscopic divalent free currents (Fig. 4 b). This 4-fold activation may correspond to the current seen in Jurkat cells by Kerschbaum and Cahalan (11Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar) in the absence of external divalent cations, but with Mg2+ present in the cytoplasm. However, in the RBL-2H3 cells no larger, transient inward current was observed. Similar kinetics for RBL-1 cells have been reported by Fiero et al. (22Fierro L. Lund P.-E. Parekh A.B. Pflügers Arch. 2000; 440: 580-587PubMed Google Scholar). Following this 4-fold jump, which was only seen in the preactivated cells, a slower activation to the full, maximally conducting state was observed, and the kinetics of this process were similar in control and preactivated cells. Likewise, the appearance of single-channel openings following excision into divalent cation-free solutions occurred with a similar delay when stores were previously emptied (Fig. 4 c). Taken together, these results demonstrate that the delay time for whole cell current activation by divalent cation-free solutions, as well as the delay time for channel activation following excision into divalent cation-free solutions does not appear to depend on depletion of Ca2+-stores. However, they do not rule out a requirement for initial store depletion, followed by a subsequent amplification step, which is rate-limiting. Nonetheless, the mechanism by which observable single-channel activation occurs appears to be at least somewhat distinct from the mechanism of activation of Icrac by store depletion. Note, however, that activation of an intermediate activity state of the channels, similar to that seen with low divalent cations outside and with Mg2+ inside (11Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar, 22Fierro L. Lund P.-E. Parekh A.B. Pflügers Arch. 2000; 440: 580-587PubMed Google Scholar), did appear to be hastened by prior depletion of Ca2+-stores. The significance of this observation will be considered subsequently under "Discussion." To further address the issue of whether the channels activated by divalent cation-free solutions represent CRAC channels, we next examined the modulation of these channels by internal divalent cations and by pH to compare their behavior which was shown previously for CRAC channels. Krause et al. (23Krause E. Schmid A. Gonzalez A. Schulz I. J. Biol. Chem. 1999; 274: 36957-36962Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) reported from whole cell studies in RBL-1 cells, that Icrac activation changed from spontaneous to storeoperated mode, if the cytosolic [Ca2+] is elevated to more than 50 nm. The spontaneous Icrac was inactivated at a resting cytosolic [Ca2+] of 105 nm. On the basis of their finding, after reaching full channel activity in the inside-out mode, we perfused the bath with intracellular solution containing 100 nm free Ca2+ (Fig.5 a). This concentration completely inhibited channel activity in 50.5 ± 9.5 s in 4 patches, while in 3 other patches channel activity was unaffected. In the 4 patches in which channel activity was inhibited, restoration of low Ca2+ conditions reversed the inhibition in only 1 of the 4 experiments. The inhibitory effect of Ca2+ was rather specific and not due to inhibition of monovalent currents by divalent ions per se, because similarly low concentrations of free Mg2+ failed to inhibit. However, if 100 μmMg2+ or more was added to the inner surface of the active channels, inhibition developed in 13.4 ± 3.1 s (12 out of 12 patches) (Fig. 5 b). The activity was restored by divalent free buffers (not shown). In both cases, as the inhibition developed, single channel conductance was unaffected indicating that the divalent cations suppress open probability. In the whole cell mode in Jurkat T-lymphocytes, the inactivation of Na+ current by intracellular Mg2+ could be reduced by increasing the intracellular pH (14Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 1998; 111: 521-537Crossref PubMed Scopus (85) Google Scholar). The experiment in Fig.5 c demonstrates a similar effect of increasing pH for the channels in excised patches. After inhibition by 100 μmMg2+ (pH 7.2), channel activity could be rescued by increasing the pH in the perfusion solution to pH 8.2 (4 out of 7 cells). The time needed for complete reversal in the cells that responded to the pH change was 42 ± 10 s. We also carried out experiments similar to those described by Kerschbaum and Cahalan (14Kerschbaum H.H. Cahalan M.D. J. Gen. Physiol. 1998; 111: 521-537Crossref PubMed Scopus (85) Google Scholar) in which the channels were inhibited by addition of a much higher concentration of Mg2+ (2.4 mm). Channel activity was blocked in 7 of 7 experiments, but in three attempts there was no recovery on shifting to pH 8.0 (not shown). From the experiments depicted in Fig. 5, some interesting observations can be made. First, the results provide evidence that the single-channels are the channels underlying the large whole cell current in divalent cation-free solutions. Second, the inhibition of the channels with Ca2+ and Mg2+ concentrations in the physiological range explains the inability to detect the same channels in the cell-attached mode. In the cell-attached mode, neither addition of 1 μm thapsigargin (n = 14) nor addition of 500 nm ionomycin (n = 13) evoked detectable single-channel activity. Note that under these conditions, whole cell currents (Icrac, pA magnitude) can be seen indicating that single channels must be opening; but these channels are not detectable because in the presence of Ca2+ or Mg2+ the single channel conductance and/or frequency of opening is below the level of detection. Third, the ability of low, physiological concentrations of Ca2+ to inhibit the channels is consistent with their identity as CRAC channels. Recent reports have supported a conformational coupling model for SOC activation, whereby IP3 receptors in the endoplasmic reticulum sense the Ca2+ filling status of the stores and transfer this information via direct coupling to SOCs in the plasma membrane (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (579) Google Scholar, 6Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1049) Google Scholar, 7Zubov A.I. Kaznacheeva E.V. Alexeeno V.A. Kiselyov K. Muallem S. Mozhayeva G. J. Biol. Chem. 1999; 274: 25983-25985Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Ma et al. (16Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (533) Google Scholar) concluded from their studies, using the membrane permeant IP3 receptor inhibitor 2-APB that IP3 receptors are essential not only for opening of SOCs, but also for maintaining their activation. On this basis, we examined the effects of 2-APB on macroscopic currents, and on channel activity in excised patches. The results shown before in Fig. 2 demonstrate that no added IP3 or added IP3 receptors are necessary to maintain activity of CRAC channels in excised patches under divalent cation-free conditions. This is in contrast to previous reports demonstrating that Trp-3 channel activity (24Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (561) Google Scholar) and store-operated channel activity in A431 cells (7Zubov A.I. Kaznacheeva E.V. Alexeeno V.A. Kiselyov K. Muallem S. Mozhayeva G. J. Biol. Chem. 1999; 274: 25983-25985Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) were rapidly lost in the absence of added IP3 and IP3 receptors. This may indicate that once activated under divalent-free conditions, CRAC channel activity is independent of Ca2+-store depletion and coupling to IP3 receptors (although we cannot unequivocally rule out the stable retention of fragments of endoplasmic reticulum and IP3 receptors). Thus, we were initially not surprised to find that addition of 100 μm 2-APB to the medium surrounding activated channels in excised patches in most instances failed to inhibit channel activity (for example, Fig.6 d). However, with this protocol 2-APB is added to the cytoplasmic side of the plasma membrane, while in previous studies of Ma et al. (16Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (533) Google Scholar), the drug was always added to the outside. Experiments illustrated in Fig. 6,a and b, show that in the whole cell configuration, with either Ca2+ or Na+ as charge carrier, 2-APB blocked the current completely when applied externally, but had significantly less effect when applied in the pipette. Finally, although 2-APB blocked channel activity when applied to the cytoplasmic side of excised patches in only 2 of 11 experiments (Fig. 6 d), if included in the patch pipette, channel activation did not occur in any of 10 separate experiments (Fig.6 c). These results suggest that 2-APB may be acting as a direct blocker of SOCs, rather than as an IP3 receptor antagonist as supposed in earlier studies. However, because of the protocols used, we considered the possibility that once the channels were activated by divalent cation-free solutions, they would lose their sensitivity to the effects of 2-A
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