Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors

Abstract

1. The effects of the IP₃-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca²⁺ release-activated Ca²⁺ current (I_CRAC) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined.

2. 2-APB elicited both stimulatory and inhibitory effects on Ca²⁺ influx through CRAC channels. At concentrations of 1–5 μm, 2-APB enhanced Ca²⁺ entry in intact cells and increased I_CRAC amplitude by up to fivefold. At levels ≥ 10 μm, 2-APB caused a transient enhancement of I_CRAC followed by inhibition.

3. 2-APB altered the kinetics of fast Ca²⁺-dependent inactivation of I_CRAC. At concentrations of 1–5 μm, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 μm) reduced or completely blocked inactivation.

4. 2-APB inhibited Ca²⁺ efflux from mitochondria.

5. 2-APB inhibited I_CRAC more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action.

6. Neither I_CRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 μg ml⁻¹ heparin.

7. I_CRAC is present in wild-type as well as mutant DT40 B cells lacking all three IP₃ receptor isoforms. 2-APB also potentiates and inhibits I_CRAC in both cell types, indicating that 2-APB exerts its effects independently of IP₃ receptors.

8. Our results show that CRAC channel activation does not require physical interaction with IP₃ receptors as proposed in the conformational coupling model. Potentiation of I_CRAC by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) activates Ca²⁺ entry across the plasma membrane in a variety of cell types, a process known as store-operated Ca²⁺ entry. Imaging and patch-clamp studies indicate that the store-operated Ca²⁺ entry mechanism exists in a wide range of tissues (Parekh & Penner, 1997; Putney, 1997; Lewis, 1999). Nevertheless, detailed information about the properties of the channels underlying store-operated Ca²⁺ entry is available only for a few cell types such as Jurkat T cells, mast cells and RBL cells. The store-operated current in these cells is highly selective for Ca²⁺ and is called the calcium release-activated Ca²⁺ current, or I_CRAC. The hallmarks of I_CRAC include an extremely high selectivity for Ca²⁺ over monovalent cations, an extremely low apparent unitary conductance for Ca²⁺, voltage-independent gating, inward rectification and multiple modes of inactivation by Ca²⁺ (reviewed by Parekh & Penner, 1997; Lewis, 1999). CRAC channels play essential roles in controlling gene expression during the activation of T cells by antigen (Partiseti et al. 1994; Fanger et al. 1995) and in triggering secretion of inflammatory mediators from mast cells stimulated by allergens (Zhang & McCloskey, 1995).

Despite the wealth of electrophysiological data on the properties of I_CRAC, neither the mechanism(s) that links store depletion to activation of the current nor the molecular identity of the channels is well understood. Hypotheses presented to explain the mechanism of activation of I_CRAC by store depletion have included activation by calcium influx factor, vesicle-mediated insertion of active channels in the membrane and physical coupling with IP₃ receptors in the ER membrane (for reviews, see Putney & McKay, 1999; Prakriya & Lewis, 2001a). The last hypothesis, known as the conformational coupling hypothesis (Berridge, 1995), has received support from observations that activation of Ca²⁺ entry requires close apposition of the ER, where IP₃ receptors are localized, and the plasma membrane (Boulay et al. 1999; Patterson et al. 1999; Ma et al. 2000). Further evidence comes from reports of binding and functional interactions between transient receptor potential (TRP)3 channels and IP₃ receptors (Kiselyov et al. 1998, 1999a; Boulay et al. 1999). Although the evidence that trp3 encodes a store-operated channel is controversial (Zitt et al. 1997; Hofmann et al. 1999; Ma et al. 2000), it has been reported that an IP₃ receptor antagonist, 2-aminoethyldiphenyl borate (2-APB), blocks not only TRP3-mediated but also native store-operated Ca²⁺ influx in HEK cells (Ma et al. 2000).

2-APB has been described as a non-competitive antagonist of the IP₃ receptor and has been shown to inhibit IP₃ receptor-induced [Ca²⁺]_i elevations in a variety of cell types (Sugawara et al. 1997; Ma et al. 2000). Inhibition of store-operated Ca²⁺ entry by 2-APB has been taken as evidence for a role of IP₃ receptors in the activation of store-operated channels and therefore as proof for conformational coupling between store-operated channels and IP₃ receptors (Ma et al. 2000; van Rossum et al. 2000). However, 2-APB is not yet well characterized in terms of its possible sites of action, and more recent work suggests that 2-APB may inhibit I_CRAC in RBL cells independently of the IP₃ receptor (Braun et al. 2001). A more thorough characterization of the effects of 2-APB on I_CRAC and other Ca²⁺ transport pathways is needed to test the potential utility of this compound in studying the activation and regulation of store-operated channels such as the CRAC channel.

To explore the possible role of IP₃ receptors in the induction and maintenance of I_CRAC, we examined the effects of 2-APB in Jurkat T cells, DT40 B cells and RBL cells. We found that 2-APB has complex effects on I_CRAC. At low concentrations (≤ 5 μm), 2-APB enhances the size of I_CRAC and speeds up fast Ca²⁺-dependent inactivation of the channels. At higher concentrations, 2-APB inhibits I_CRAC while also removing Ca²⁺-dependent inactivation. These complex effects on I_CRAC also occur in mutant DT40 cells that lack IP₃ receptors. Thus, our results indicate that 2-APB elicits multiple effects on I_CRAC gating by a mechanism that is independent of IP₃ receptors. These results have been reported previously in abstract form (Prakriya & Lewis, 2001b).

METHODS

Cells

Jurkat T cells (Jurkat E6–1 cell line) were grown in medium consisting of RPMI 1640 supplemented with 10 % fetal calf serum (FCS), 2 mm glutamine and penicillin-streptomycin. DT40 B cells and DT40 IP₃ receptor triple-knockout cells (RIKEN Cell Bank, Ibaraki, Japan) were maintained in a medium containing RPMI 1640 supplemented with 10 % FCS, 5 % chicken serum, 4 mm glutamine, 50 μmβ-mercaptoethanol and penicillin-streptomycin. RBL-2H3 cells were grown in DMEM supplemented with 10 % FCS, 4 mm glutamine and penicillin-streptomycin. All cells were maintained in log-phase growth at 37 °C in 6 % CO₂.

Solutions and chemicals

The extracellular solution contained (mm): 155 NaCl, 4.5 KCl, 1 MgCl₂, 10 d-glucose and 5 Na-Hepes (pH 7.4) with 2 mm CaCl₂ in Ca²⁺ imaging experiments, and 20 mm CaCl₂ or 20 mm BaCl₂ in patch-clamp experiments. Ca²⁺-free solutions contained 1 mm EGTA and 3 mm MgCl₂. The intracellular (pipette) solution contained (mm): 150 caesium methanesulfonate, 10 Cs-Hepes, 10 EGTA and 3 MgCl₂ (pH 7.3). In some experiments, a weakly buffered internal solution containing 1.2 mm EGTA was used. To isolate I_CRAC from inward rectifier K⁺ currents in RBL-2H3 cells, 10 mm CsCl₂ was added to the standard extracellular solution. In experiments involving caged IP₃, 15 μm NPE-IP₃ (Calbiochem) was included in the patch pipette, and the internal solution contained 1.2 mm EGTA + 0.705 mm CaCl₂ (calculated free [Ca²⁺]= 150 nm) to prevent store depletion prior to IP₃ release.

2-APB was the kind gift of Dr K. Mikoshiba (Tokyo University, Japan). In some experiments, 2-APB purchased from Sigma Chemical Co. (St Louis, MO, USA) was used; the drugs from both sources had identical effects on I_CRAC. Stock solutions of 2-APB, thapsigargin (TG; Sigma) and oligomycin (Sigma) were prepared in DMSO at concentrations of 20, 1 and 2 mm respectively. Antimycin A1 (Sigma) was dissolved in ethanol at 2 mm. The drugs were diluted to the concentrations indicated in the figure legends and applied to the cells using a multibarrel local perfusion pipette with a common delivery port. The solution exchange time was measured to be < 2 s, based on the rate at which the K⁺ current reversal potential changed when the external solution was switched from 2 mm K⁺ to 150 mm K⁺.

[Ca²⁺]_i imaging

Cells were loaded for 15 min at 37 °C with 1 μm fura-2 AM (Molecular Probes, OR, USA) dissolved in Jurkat medium, then were washed with Jurkat or DT40 medium and stored in the dark until ready for use. Fura-2-loaded cells were plated on glass coverslips coated with poly-d-lysine and were preincubated for several minutes in an external solution containing 0.5 mm Ca²⁺. Coverslips were mounted on the stage of a Zeiss Axiovert 35 microscope equipped with a ×40 Achrostigmat objective (NA 1.3). Imaging protocols and equipment were as described previously (Hoth et al. 1997). Briefly, cells were alternately illuminated at 350 and 380 nm using band-pass filters (Chroma Technology) and the fluorescence emissions at wavelengths greater than 480 nm were captured with an intensified CCD camera (Hamamatsu, Japan) and digitized and analysed using a VideoProbe imaging system (ETM Systems, Irvine, CA, USA). The 350 nm/ 380 nm ratio images were recorded at intervals of 5 s. [Ca²⁺]_i was calculated from the relationship [Ca²⁺]_i=K(R – R_min)/(R_max–R), where the values of K, R_min and R_max were determined from an in situ calibration of fura-2 in Jurkat T cells loaded by intracellular dialysis (Lewis & Cahalan, 1989). R_min and R_max are the fluorescence ratios determined with internal solutions containing 10 mm EGTA and 10 mm CaCl₂, respectively; K was determined using an internal solution containing 180 nm free [Ca²⁺]_i.

Patch-clamp recording

Patch-clamp experiments were conducted in the standard whole-cell recording configuration using recording electrodes that were pulled from 100 μl pipettes, coated with Sylgard and fire polished to a final resistance of 2–5 MΩ. Data were collected with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA, USA) interfaced to an ITC-16 input/output board (Instrutech, Port Washington, NY, USA) and a Macintosh G3 computer. Stimulation, data acquisition and analysis were performed using in-house software developed on the Igor Pro platform (Wavemetrics, Lake Oswego, OR, USA). The command voltage protocol usually consisted of a 100 ms step from the holding potential to −100 mV followed immediately by a 100 ms ramp from −100 to +60 mV, applied every 1–2 s. The holding potential was +30 mV unless otherwise indicated. Current was sampled at a rate of 5 kHz and filtered at 500 Hz offline. All data were corrected for leak currents that were collected using the same voltage stimulus in a Ca²⁺-free bath solution, after I_CRAC induction and before data collection. Unless otherwise indicated, all average results are presented as the mean value ±s.e.m.

Two methods were typically employed to deplete intracellular Ca²⁺ stores and activate I_CRAC. With the first method, stores were passively depleted by dialysing the cells with an internal recording solution containing either 10 or 1.2 mm EGTA. With the second method, cells were preincubated in the recording chamber with Ca²⁺-free Ringer solution containing 1 μm TG for 5–10 min before seal formation. In the experiments of Fig. 9C, I_CRAC was activated by the release of caged IP₃; NPE-IP₃ was photolysed by a 10 s exposure to unattenuated light from a xenon light source (Lambda-10, Sutter Instruments) passed through a 360 nm interference filter (Chroma Technology).

Figure 9. Heparin does not interfere with the activation of I_CRAC by passive store depletion or with the effects of 2-APB.

In all experiments except as noted, 500 μg ml⁻¹ heparin was included in the recording pipette, and I_CRAC was induced through passive depletion of internal stores by 10 mm EGTA in the patch pipette. A and B, heparin affects neither the rate of I_CRAC activation (A) nor its maximal amplitude at steady state (B). The time to steady state was measured as the time to reach 95 % of the maximal current amplitude. Current amplitude was normalized to cell capacitance (average C_m= 9.95 pF). Heparin-treated cells (n = 6) are compared with matched control cells (n = 7). C, heparin loading via the patch pipette effectively inhibited IP₃ receptors. In control Jurkat cells lacking heparin, I_CRAC was activated by a 10 s UV flash to release caged IP₃ (15 μm). With 500 μg ml⁻¹ heparin in the pipette, uncaging IP₃ failed to activate I_CRAC. Results are representative of four cells loaded with caged IP₃ alone and three cells with caged IP₃ and heparin. D, 40 μm 2-APB first potentiated and then inhibited I_CRAC in a Jurkat cell loaded with heparin via the patch pipette. Shown here are the peak currents during steps to −100 mV from +30 mV. E, summary of the potentiating effects of 5 μm 2-APB in heparin-treated (n = 6) and matched control (n = 5) cells.

RESULTS

Inhibition of store-operated Ca²⁺ influx and mitochondrial Ca²⁺ efflux by 2-APB

The effects of 2-APB on Ca²⁺ influx through CRAC channels were first examined in Ca²⁺ imaging experiments using fura-2-loaded Jurkat T cells. TG (1 μm), a membrane-permeant inhibitor of the SERCA Ca²⁺-ATPase in the ER (Thastrup et al. 1989), was applied for ∼500 s in a Ca²⁺-free solution to deplete intracellular Ca²⁺ stores and activate CRAC channels (Fig. 1). Subsequent restoration of Ca²⁺ to the bath produces a rapid elevation of [Ca²⁺]_i due to influx through open CRAC channels, followed by a gradual decline to a high steady-state [Ca²⁺]_i plateau, as the rates of Ca²⁺ influx and Ca²⁺ clearance reach a new balance (Hoth et al. 1997). As shown in Fig. 1A, application of 40 μm 2-APB rapidly abolished the [Ca²⁺]_i elevation and brought [Ca²⁺]_i back to baseline within 120 s, similar to its reported effects on store-operated Ca²⁺ entry in other cell types (Ma et al. 2000; van Rossum et al. 2000). In addition, application of 40 μm 2-APB in a Ca²⁺-free solution eliminated the subsequent rise in [Ca²⁺]_i upon Ca²⁺ re-addition (data not shown), indicating that the inhibitory effect required neither external Ca²⁺ nor elevated [Ca²⁺]_i. Thus, these results confirmed that 2-APB very effectively blocks [Ca²⁺]_i elevations produced by Ca²⁺ influx through CRAC channels.

Figure 1. Effects of 2-APB on Ca²⁺ entry through CRAC channels and on Ca²⁺ release from mitochondria.

In each panel, TG (1 μm; stippled bar) was applied in Ca²⁺-free Ringer solution to irreversibly deplete stores and activate CRAC channels in Jurkat T cells. Subsequent addition of 2 mm Ca²⁺ to the bath (filled bar) elicited a rise in [Ca²⁺]_i. Each trace represents the average of 150–200 cells. A, a high concentration of 2-APB (40 μm; hatched bar) inhibited Ca²⁺ influx through CRAC channels. The arrow marks a transient enhancement of [Ca²⁺]_i prior to inhibition. B, 2-APB inhibits Ca²⁺ release by mitochondria. After Ca²⁺ influx through CRAC channels was terminated by removing extracellular Ca²⁺, [Ca²⁺]_i fell to a low elevated plateau that reflected the export of Ca²⁺ from mitochondria (dashed line). Addition of 50 μm 2-APB to the Ca²⁺-free solution rapidly and reversibly reduced [Ca²⁺]_i to baseline due to inhibition of mitochondrial Ca²⁺ export. A lower concentration of 2-APB (5 μm) produced a smaller effect. C, a low concentration of 2-APB (10 μm) did not elevate [Ca²⁺]_i in resting cells before internal stores had been depleted by TG. However, after I_CRAC activation, 2-APB enhanced the [Ca²⁺]_i rise resulting from influx through CRAC channels.

High doses of 2-APB reduced [Ca²⁺]_i relatively rapidly to baseline values, even when preceded by a long, large [Ca²⁺]_i elevation (Fig. 1A). Previous work has shown that mitochondria take up a substantial amount of Ca²⁺ during such extended periods of influx, such that when influx is terminated (e.g. by removal of extracellular Ca²⁺), [Ca²⁺]_i falls to a low plateau value that reflects a balance between Ca²⁺ pumping across the plasma membrane and Ca²⁺ efflux from the loaded mitochondria (marked with a dashed line in Fig. 1B; Hoth et al. 1997). This plateau declines with a time constant of hundreds of seconds as the mitochondria slowly unload their Ca²⁺. The lack of this low [Ca²⁺]_i plateau in the presence of 2-APB suggests that the drug might prevent Ca²⁺ release from mitochondria. To test this idea, we applied 2-APB in Ca²⁺-free solution after a period of prolonged [Ca²⁺]_i elevation. Addition of 50 μm 2-APB during the mitochondria-dependent Ca²⁺ plateau immediately caused [Ca²⁺]_i to decline back to baseline, consistent with an acute inhibition of mitochondrial Ca²⁺ export (Fig. 1B). In principle, this result could also be produced by an increased rate of Ca²⁺ extrusion across the plasma membrane; however, 2-APB did not alter the initial rate of [Ca²⁺]_i decline when ${\text{Ca}}_0^{2+}$ was first removed (data not shown), demonstrating that the drug does not detectably affect Ca²⁺ pumps in the plasma membrane. Washout of the drug allowed Ca²⁺ to recover to the plateau level, often with a small overshoot. This behaviour would be expected from the resumption of mitochondrial export and a delayed approach to a new equilibrium between mitochondrial Ca²⁺ export and Ca²⁺ clearance across the plasma membrane. A lower concentration of 2-APB (5 μm) caused less-complete inhibition of mitochondrial export (Fig. 1B). Because a Na⁺-Ca²⁺ exchanger in the mitochondrial inner membrane is believed to be responsible for carrying out a significant proportion of Ca²⁺ export (Gunter et al. 2000), we speculate that 2-APB may inhibit the mitochondrial Na⁺-Ca²⁺ exchanger. We did not explore the mechanism of this mitochondrial effect further, but instead focused on the effects of 2-APB on Ca²⁺ influx.

Potentiation of store-operated Ca²⁺ entry by 2-APB

An unexpected observation was that the inhibition of Ca²⁺ influx was always preceded by a transient elevation of [Ca²⁺]_i (Fig. 1A, arrow). When the concentration of 2-APB was reduced to 10 μm, the [Ca²⁺]_i rise induced by 2-APB became much more pronounced and sustained (Fig. 1C). Interestingly, this potentiation of Ca²⁺ influx occurred only after store depletion; no effects were seen after application of dilute 2-APB to resting T cells (Fig. 1C). These results indicate that, in addition to its known ability to inhibit store-operated Ca²⁺ influx, 2-APB can also potentiate Ca²⁺ entry in Jurkat T cells.

The progression from potentiation to inhibition of Ca²⁺ entry as 2-APB concentration is increased is seen most clearly in the dose-response experiment of Fig. 2. Whereas low concentrations of 2-APB (≤ 5 μm) elicited [Ca²⁺]_i elevations that were largely sustained during the 400 s of drug application, high concentrations (≥ 10 μm) produced a transient rise followed by a progressive fall in [Ca²⁺]_i. The rate of both the rise and the decay of [Ca²⁺]_i were accelerated as the concentration of 2-APB was increased.

Figure 2. Potentiation and inhibition of Ca²⁺ entry by 2-APB are concentration dependent.

Six representative experiments with increasing concentrations of 2-APB are superimposed. In each experiment, TG followed by re-addition of 2 mm Ca²⁺ was used to elicit a high [Ca²⁺]_i plateau. As [2-APB] was increased, sustained potentiation of the [Ca²⁺]_i rise gave way to transient potentiation followed by inhibition. Data were normalized to the [Ca²⁺]_i measured immediately before addition of 2-APB, to allow better comparison between the different experiments. The average [Ca²⁺]_i at this time was 1342 ± 20 nm (mean ±s.e.m.).

Potentiation and blockade of I_CRAC by 2-APB

Several mechanisms could in principle account for the effects of 2-APB on [Ca²⁺]_i in the imaging experiments shown in Figs 1 and 2. These include potentiation and block of CRAC channels as well as mechanisms unrelated to CRAC channels, such as alteration of the membrane potential and driving force for Ca²⁺ entry by modulation of K⁺ channels. To distinguish between these possibilities, we directly examined the effects of 2-APB on I_CRAC in whole-cell recordings. I_CRAC was induced in these experiments by passively depleting internal Ca²⁺ stores through intracellular dialysis with 10 mm EGTA in the patch pipette (Hoth & Penner, 1992; Zweifach & Lewis, 1993), and the peak amplitude of I_CRAC was measured during repetitive hyperpolarizing pulses to −100 mV.

The effects of 2-APB on the current paralleled those on [Ca²⁺]_i. Thus, 5 μm 2-APB evoked a large sustained increase in the amplitude of the inward current at −100 mV (Fig. 3A, top trace), whilst 20 μm 2-APB induced an initial increase in current followed by a slow decline in I_CRAC amplitude (Fig. 3A, middle trace). Increasing the 2-APB concentration to 40 μm increased the rate of potentiation and the subsequent blockade of I_CRAC (Fig. 3A, bottom trace). The decay of I_CRAC followed an approximately single-exponential time course. No effects were seen in control experiments in which the corresponding concentration of DMSO was applied (data not shown). Therefore, these results suggest that whereas high concentrations of 2-APB block I_CRAC, low concentrations activate an additional inward current.

Figure 3. Potentiation and inhibition of I_CRAC by 2-APB.

A, effects of 2-APB on I_CRAC. Current was activated by depleting Ca²⁺ stores passively with 10 mm EGTA in the recording pipette. 2-APB at 5 μm (top) enhanced I_CRAC amplitude over the duration of drug application, whereas 20 μm (middle) and 40 μm 2-APB (bottom) initially enhanced, then inhibited, I_CRAC. Shown here are the peak Ca²⁺ currents elicited during 200 ms steps to −100 mV from a holding potential of +10 mV. B–E, evidence that the additional inward current activated by 2-APB is I_CRAC. B, the current-voltage relation of the inward current activated by store depletion before and after 2-APB (10 μm). C, 2-APB (10 μm) did not activate inward current in Ca²⁺-free Ringer solution. I_CRAC was induced passively as described above. D, the inward current activated by 2-APB (10 μm) is blocked by extracellular La³⁺ (1.5 μm). E, 2-APB (5 μm) does not elicit inward current before I_CRAC is activated. After the delayed induction of I_CRAC by intracellular EGTA (1.2 mm), application of 2-APB potentiated I_CRAC robustly.

Several lines of evidence indicate that the additional inward current activated by 2-APB is I_CRAC itself rather than an unrelated current. First, the I–V profile and prominent inward rectification of the current in 2-APB were identical to that of I_CRAC (Fig. 3B). Second, consistent with the high Ca²⁺ selectivity of I_CRAC, the inward current was absent in nominally Ca²⁺-free external solutions (Fig. 3C). Third, the 2-APB-induced current was completely blocked by low concentrations of La³⁺ (1.5 μm; Fig. 3D) and SKF 96365 (not shown), two known inhibitors of I_CRAC (Franzius et al. 1994; Aussel et al. 1996). Finally, 2-APB only induced inward current subsequent to store depletion and activation of I_CRAC by EGTA-containing internal solutions. In the experiment shown in Fig. 3E, brief application of 2-APB (5 μm) before the passive induction of I_CRAC had no effect, but after I_CRAC was activated, the same dose caused robust potentiation. Taken together with the evidence that a low dose of 2-APB elevated [Ca²⁺]_i only after stores were depleted (Fig. 1C), these data indicate that the transient inward current activated by 2-APB is I_CRAC itself. Thus, we conclude that 2-APB elicits both excitatory and inhibitory effects on I_CRAC.

The potentiating and inhibitory effects of 2-APB on I_CRAC differed in two ways. First, although potentiation of I_CRAC was rapidly reversible (Fig. 3A, top trace; average time constant of recovery, τ_rec= 6.2 ± 1.3 s, n = 6), inhibition of I_CRAC in Jurkat cells reversed extremely slowly following removal of the drug (Fig. 3A, bottom trace and Fig. 6A). In four cells exposed to 40 μm 2-APB, washout of the drug caused the current to recover by an average of 22 % after 160 s, suggesting a τ_rec of > 600 s. A second difference was in the concentration dependence of the two effects. Current potentiation was evident at lower drug concentrations than those that inhibited I_CRAC. Unfortunately, an overlap in the effective concentrations for potentiation and inhibition complicated efforts to estimate separately the K_1/2 values for the two processes. The concentration dependence of I_CRAC enhancement measured directly from the peak current amplitudes before and after exposure to 2-APB is shown in Fig. 4A (filled circles). Potentiation peaked around 5 μm, then decreased with further increases in 2-APB concentration, presumably because of increasing current inhibition. To attempt to correct for the effect of inhibition and estimate the true extent of potentiation by 2-APB at these concentrations, we fitted a single-exponential function to the current decay induced by 2-APB and extrapolated the current amplitude back to the time the drug was first applied (Fig. 4A, open circles). The estimated potentiation at different concentrations of 2-APB was then fitted with a Hill equation, yielding an apparent K_1/2 of ∼3 μm for potentiation. By contrast, the apparent K_1/2 for I_CRAC blockade as measured by inhibition of the peak current was ∼10 μm (Fig. 4B). This sensitivity of CRAC channels to inhibition by 2-APB is identical to that reported by Ma et al. (2000) for inhibition of TRP3-mediated Sr²⁺ entry in transfected HEK 293 cells. However, it is significantly lower than the IC₅₀ for inhibition of Ca²⁺ release from IP₃ receptors (25–42 μm; Maruyama et al. 1997; Ma et al. 2000).

Figure 6. High concentrations of 2-APB eliminate fast inactivation of I_CRAC.

A, plot of peak I_CRAC (•) and the extent of inactivation (○) during pulses to −100 mV before and after exposure to 40 μm 2-APB (hatched bar). The extent of inactivation was measured at the end of the 250 ms test pulses (I_t= 250 ms; see legend to Fig. 5). Examples of currents at various time points are shown below to illustrate the removal of fast inactivation. B, inhibition of I_CRAC and removal of fast inactivation occur at a similar rate. The time courses of I_CRAC inhibition and reduction in the extent of inactivation were fitted with single-exponential functions and the time constants from the resulting fits are shown (40 μm 2-APB; n = 5). C, dose dependence of the removal of inactivation by 2-APB. The extent of inactivation was measured 100 ms after steps to −100 mV, at a time when the effects of 2-APB had reached steady state. Each point represents the mean of three to six cells.

Figure 4. Concentration dependence of potentiation and inhibition of I_CRAC by 2-APB.

A, extent of I_CRAC potentiation as a function of 2-APB concentration. Filled circles show peak currents measured from experiments similar to those in Fig. 3. To correct for the overlap of potentiation and inhibition at 2-APB concentrations ≥ 10 μm, peak currents were also estimated by back-extrapolation (open circles; see text). The dashed line indicates a fit to the Hill equation Y =Y_max/(1 + ([2-APB]/K_1/2)^nH), where Y_max is the maximal potentiation, K_1/2 is the concentration that yields half-maximal potentiation, and n_H is the apparent cooperativity of the process. The best fit was obtained with K_1/2= 3.1 μm and n_H= 4.1. B, degree of I_CRAC inhibition as a function of 2-APB concentration. Inhibition was calculated from the steady-state current level relative to the peak current after addition of 2-APB. The dashed line is a fit of the Hill equation, with K_1/2= 9.6 μm and n_H= 4.2. Experimental conditions were as described for Fig. 3; holding potential =+30 mV. In A and B, each point represents the mean ±s.e.m. of three to five cells.

Low concentrations of 2-APB enhance fast inactivation of I_CRAC

Ca²⁺ entering through CRAC channels causes them to inactivate over tens of milliseconds during large hyperpolarizing pulses (Hoth & Penner, 1993; Zweifach & Lewis, 1995b; Fierro & Parekh, 1999a). Fast inactivation is a biexponential process and is believed to result from Ca²⁺ binding to multiple intracellular sites located within several nanometers of the channel pore (Zweifach & Lewis, 1995b). Interestingly, 2-APB altered fast inactivation in parallel with its effects on the amplitude of I_CRAC. At low concentrations that caused potentiation, 2-APB increased the rate and extent of fast inactivation (Fig. 5). Enhancement of fast inactivation by dilute 2-APB was observed across a wide range of test potentials, where the predominant effect was to accelerate the fastest exponential component while increasing its amplitude. As shown in Fig. 5A, this effect developed with a similar time course to the increase in peak I_CRAC (potentiation), suggesting that the two phenomena may be related. At long times (> 150 s), the inactivation rate actually decreased (not shown), perhaps due to the slow removal of inactivation at these low 2-APB concentrations (see below). There are several possible explanations for the speeding of I_CRAC inactivation by dilute 2-APB; a likely possibility is that by increasing the activity of CRAC channels, 2-APB enhances the accumulation of Ca²⁺ at the inactivation sites. This and other alternative mechanisms are discussed below (see Discussion).

Figure 5. Low concentrations of 2-APB accelerate fast inactivation of I_CRAC.

A, peak I_CRAC (•) and the extent of inactivation (○) measured during steps to −100 mV. To monitor changes in the speed of inactivation, the extent of inactivation was measured as (I_peak–I_t)/I_peak, where I_peak is the peak current and I_t is the current measured 50 ms after each step to −100 mV. Addition of 5 μm 2-APB enhanced the peak current amplitude and increased the extent of inactivation. B, the time course of inactivation in the presence and absence of 2-APB. Traces on the left show the averages of five leak-corrected responses to hyperpolarizations given in A (bars in A) in 20 mm ${\text{Ca}}_0^{2+}$ with or without 5 μm 2-APB. The right plot shows the same traces normalized to their peak current. Note that the rate of current decay was enhanced in the presence of 2-APB. Holding potential =+30 mV.

High concentrations of 2-APB remove fast inactivation of I_CRAC

In addition to inhibiting I_CRAC, a dramatic effect of high concentrations of 2-APB was to eliminate fast inactivation. In the example shown in Fig. 6A, 40 μm 2-APB evoked a rapid transient increase in I_CRAC followed by a slow and complete inhibition of the current over the next 40 s. As can be seen in the lower traces, the extent of inactivation during brief voltage pulses from +10 to −100 mV decreased over the duration of exposure to the drug. The removal of inactivation (open circles) appeared to occur in parallel with inhibition of the peak current (filled circles). The exponential time constants for the two effects were similar (Fig. 6B; n = 5 cells), suggesting that the two could be coupled. The simplest hypothesis would be that the inhibition of current through CRAC channels reduces inactivation by lessening the accumulation of intracellular Ca²⁺. This seems unlikely, however. In Fig. 6A, traces a (before 2-APB was applied) and c (collected after 2-APB had potentiated and inhibited I_CRAC) show identical peak current amplitudes even though inactivation was almost completely absent in c. Hence, while the removal of inactivation and drug-induced inhibition of the current may be related (see Discussion), inactivation did not disappear because of reduced Ca²⁺ flux through CRAC channels.

The removal of inactivation by 2-APB measured during drug applications lasting 150 s or longer was dose dependent, increasing in extent from 5 to 50 μm (Fig. 6C). The threshold concentration for this effect appears to be ∼5 μm, as judged from the small delayed decline in the extent of inactivation at this dose of 2-APB (see above).

The potentiation of I_CRAC by 2-APB is not due to removal of fast inactivation

Given that 2-APB was able to inhibit fast Ca²⁺-dependent inactivation of I_CRAC (Fig. 6), we considered the possibility that a reduction of steady-state inactivation might contribute to potentiation of the current. As a first test, we measured the degree of inactivation under steady-state conditions in the presence and absence of 2-APB (Fig. 7). Following a 1 s conditioning step to varying potentials, I_CRAC peak amplitude was measured in response to a step to −100 mV. I_CRAC amplitude was roughly constant following conditioning steps to +20 mV and above, but declined following more negative conditioning steps to a level of ∼40 % at −80 mV and below. A dose of 2-APB (5 μm) that caused a severalfold potentiation of I_CRAC reduced this inactivation by only ∼20 % at the most negative potentials. Therefore, it seems unlikely that the modest effect of dilute 2-APB on inactivation makes a large contribution to potentiation. This conclusion was tested further by investigating whether 2-APB could potentiate I_CRAC under conditions that prevented fast inactivation. The degree of potentiation by 2-APB was similar at holding potentials of −50 or +50 mV (Fig. 7B), even though steady-state inactivation was prominent at −50 mV but absent at +50 mV (see Fig. 7A). A third test exploited the fact that Ba²⁺ currents through CRAC channels do not show fast inactivation (Zweifach & Lewis, 1995b). 2-APB potentiated Ba²⁺ currents through CRAC channels to at least as great a degree as it did Ca²⁺ currents (Fig. 7C and D). These results show clearly that the removal of inactivation does not explain the potentiation of I_CRAC by 2-APB.

Figure 7. Potentiation of I_CRAC by 2-APB does not result from the removal of fast inactivation.

A, 2-APB alters the extent of steady-state inactivation of I_CRAC. The extent of inactivation was manipulated by applying a 1 s conditioning step to various potentials (inset) followed by a test pulse to −100 mV where I_CRAC amplitude was measured. Steady-state inactivation at each potential, Inact(V), was calculated from the relation Inact(V) = 1 − (I_cond/I_{60 mV}), where I_cond is the peak current during a test pulse to −100 mV after a conditioning step to potential V, and I_{60 mV} is the peak current at −100 mV following a conditioning step to +60 mV. Holding potential =+60 mV; n = 4 cells. B, potentiation of I_CRAC by 5 μm 2-APB is only marginally reduced at positive potentials that remove resting inactivation. Peak I_CRAC amplitude was measured during 50 ms test pulses to −100 mV from the indicated holding potential; n = 5 cells. C, potentiation of I_CRAC by 2-APB persists when Ba²⁺ is used as the charge carrier. I_CRAC was activated by internal dialysis with 10 mm EGTA. Traces show sequential recordings of current from one cell during test pulses to −100 mV in the presence of 20 mm ${\text{Ca}}_0^{2+}$ (Ca), then shortly after 20 mm Ba²⁺ was substituted for ${\text{Ca}}_0^{2+}$ (Ba), and finally after 10 μm 2-APB was applied in the presence of Ba²⁺ (Ba + 2-APB). Even though fast inactivation did not occur in ${\text{Ba}}_0^{2+}$, 2-APB induced robust potentiation. D, summary of potentiation elicited by 10 μm 2-APB in 20 mm ${\text{Ca}}_0^{2+}$ (n = 9 cells) or ${\text{Ba}}_0^{2+}$ (n = 5 cells).

Inhibition and potentiation of I_CRAC by 2-APB is not cell specific

Although 2-APB has been reported to block I_CRAC in RBL cells (Braun et al. 2001), no information exists about whether it potentiates I_CRAC in any cells other than Jurkat human T cells. Therefore, we tested the effects of low and high doses of 2-APB on I_CRAC in RBL cells and DT40 cells, a chicken pre-B cell line. TG-treated DT40 cells exhibited inwardly rectifying currents with reversal potentials > +30 mV (Fig. 8A and B). The pharmacological hallmarks of this current were also similar to I_CRAC in Jurkat T cells: it was effectively blocked by 20 μm La³⁺ as well as by 30 μm SKF 963615 (data not shown).

Figure 8. 2-APB potentiates and inhibits I_CRAC in DT40 B cells and RBL cells.

A, effects of 50 μm 2-APB on I_CRAC in a DT40 cell. I_CRAC was induced by exposing the cell to 1 μm TG for 5 min prior to seal formation; I_CRAC during pulses to −100 mV is shown. B, the current-voltage relation of Ca²⁺ currents before and after exposure to 2-APB, collected at the times shown in A. C, effects of 20 μm 2-APB on I_CRAC in an RBL cell. I_CRAC was induced by passive depletion of internal stores by 10 mm EGTA in the patch pipette. Note the smaller extent of I_CRAC enhancement by 2-APB. D, current-voltage profile of I_CRAC in the presence and absence of 2-APB, collected at the times shown in C.

The effects of 2-APB on I_CRAC in RBL and DT40 cells were similar to those observed in Jurkat T cells. In both cell types, high doses of 2-APB elicited a transient potentiation of I_CRAC, which was then followed by a slow and progressive inhibition that was largely irreversible (Fig. 8). The degree of potentiation in RBL cells tended to be somewhat smaller than that observed in either Jurkat or DT40 cells: 20 μm 2-APB elicited only a 28 ± 8 % increase in I_CRAC current amplitude in RBL cells (n = 4 cells), compared with ∼100 % in Jurkat cells (see Fig. 4A). As in Jurkat cells, the high concentration of 2-APB removed fast inactivation of I_CRAC in RBL and DT40 cells. Thus, these results indicate that the complex effects of 2-APB on CRAC channel gating were not specific to a single cell type or species.

CRAC channel activation and the effects of 2-APB are independent of IP₃ receptors

2-APB has been reported to be a selective antagonist of the IP₃ receptor (Maruyama et al. 1997). The inhibition of store-operated Ca²⁺ influx by 2-APB has therefore been attributed to the inhibition of IP₃ receptors, and has been used to support the notion that store-operated channels are activated through conformational coupling to IP₃ receptors (Ma et al. 2000). Therefore, a critical question is whether I_CRAC is dependent on IP₃ receptors, and whether the complex effects of 2-APB on I_CRAC are due to the drug acting through IP₃ receptors as opposed to other targets, possibly the CRAC channel itself. We examined this issue using three independent approaches. First, we examined the effects of heparin, an IP₃ receptor antagonist, on I_CRAC to determine whether IP₃ binding to its receptor was necessary for I_CRAC activation or the effects of 2-APB. Heparin potently inhibits IP₃ binding with a K_i of 2–10 μg ml⁻¹ (Supattapone et al. 1988; Parys et al. 1992), and previous reports have indicated that it does not affect passive induction of I_CRAC in mast and RBL cells (Hoth & Penner, 1993; Fierro & Parekh, 1999b). Consistent with these reports, we found that, in Jurkat T cells dialysed through the recording pipette with 10 mm EGTA to activate I_CRAC, heparin (500 μg ml⁻¹) had no effect on either the current amplitude or time course of induction (Fig. 9A and B). Under these conditions, heparin was clearly effective in inhibiting IP₃ receptors because it did block the rapid induction of I_CRAC by uncaging intracellular IP₃ (Fig. 9C). Importantly, intracellular heparin also failed to affect the ability of 2-APB to either potentiate or inhibit I_CRAC (Fig. 9D and E). These results suggest that the binding of background levels of IP₃ is not necessary for activation of I_CRAC through passive store depletion, nor is it required for the effects of 2-APB on I_CRAC.

Next, we investigated whether 2-APB preferentially acts upon I_CRAC from the inside or the outside of the cell. A previous report (Braun et al. 2001) showed that 2-APB inhibits I_CRAC in RBL cells more effectively when applied outside, and we found a similar result in Jurkat cells. Applying 2-APB intracellularly by including a high concentration of the drug (80 μm) in the patch pipette did not significantly inhibit either the induction or the maintenance of I_CRAC (Fig. 10A and B). In contrast, subsequent exposure of the cells to 40 μm 2-APB extracellularly caused significant inhibition (Fig. 10A). The greater potency of extracellular 2-APB appears to support the idea that the site of action is extracellular and therefore independent of IP₃ receptors. However, the interpretation of this experiment is complicated by the fact that 2-APB is membrane permeant. It is well established that lipophilic compounds that are known to block ion channels from an intracellular site often act more effectively when applied from the outside (DeCoursey, 1995). This paradox results from the fact that when membrane-permeant compounds are applied through the recording pipette, their concentration at the membrane may be lower than expected because diffusion out of the cell is faster than diffusion out of the pipette (DeCoursey, 1995). The membrane permeability and protonation state of 2-APB under our experimental conditions was not known, but a reduction of extracellular pH would have been expected to increase the protonation of 2-APB and reduce the concentration of the more membrane-permeant, non-protonated form. If the site of action were intracellular, then increased protonation of 2-APB would impair access to the site and result in a reduced effect. Therefore, we examined the effects of extracellular pH on potentiation and inhibition by bath-applied 2-APB. Reducing extracellular pH from 7.4 to 6.4 partially inhibited I_CRAC (Fig. 10C; see also Malayev & Nelson, 1995). However, the degrees of potentiation and inhibition of I_CRAC by 2-APB were identical at the two pH values (Fig. 10D). Therefore, these data support the idea that the site of action of 2-APB is extracellular rather than intracellular.

Figure 10. 2-APB may have an extracellular site of action.

A, intracellular 2-APB is ineffective at inhibiting I_CRAC. I_CRAC was activated by dialysis with 10 mm EGTA in the presence of 80 μm 2-APB in the recording pipette. Peak I_CRAC amplitudes during repeated steps to −100 mV are shown. After I_CRAC amplitude reached steady state, application of 40 μm 2-APB via the bath elicited the normal sequence of potentiation and inhibition. B, comparison of the steady-state current amplitudes of I_CRAC (normalized to cell capacitance) in cells treated with intracellular 2-APB (80 μm; n = 5 cells) and matched controls (n = 7 cells) in which I_CRAC was activated by intracellular EGTA only. C, lowering extracellular pH does not alter the ability of 2-APB to potentiate or inhibit I_CRAC. I_CRAC was activated by treatment with 1 μm TG immediately prior to seal formation. After allowing the I_CRAC amplitude to reach steady state, the external pH was lowered from 7.4 to 6.4, which dramatically reduced the current amplitude. Application of 40 μm 2-APB potentiated and inhibited I_CRAC in a manner similar to that at pH 7.4. D, summary of the extent of potentiation and inhibition of I_CRAC induced by 40 μm 2-APB at pH 7.4 (n = 5 cells) and pH 6.4 (n = 5 cells).

An extracellular site of action raises the possibility that 2-APB does not exert its effects on I_CRAC through the intracellular IP₃ receptor. To provide a more direct test of this idea, we examined the effects of 2-APB on I_CRAC in mutant DT40 cells in which all three isoforms of the IP₃ receptor (IP₃R) were knocked out by homologous recombination (Sugawara et al. 1997). These cells have been shown to lack functional type I, II and III IP₃ receptors as assayed by their failure to release Ca²⁺ in response to stimulation of phospholipase C-linked B-cell receptors or muscarinic receptors, and Northern analysis confirmed the absence of the IP₃ receptor mRNA sequence in regions including and immediately surrounding the knockout sites (Sugawara et al. 1997). In addition, recent measurements showed that, unlike their wild-type counterparts, the DT40 mutant cells bind essentially no ³H-IP₃ (Broad et al. 2001), suggesting that the amino-terminal cytoplasmic domain of the IP₃ receptor, which has been proposed to couple to store-operated channels, is also missing. Despite the lack of IP₃ receptors, store-operated Ca²⁺ entry in these cells was shown to be normal (Sugawara et al. 1997). Consistent with these results, we found that the IP₃R^−/− DT40 mutants exhibited CRAC currents that were virtually indistinguishable from those of control DT40 cells in terms of their size, I–V relationship and Ca²⁺-dependent fast inactivation (Fig. 11A–C). Thus, I_CRAC activation does not appear to require any of the known isoforms of the IP₃ receptor.

Figure 11. Effects of 2-APB are retained in mutant DT40 cells lacking IP₃ receptors.

A, I_CRAC was induced in a mutant DT40 IP₃R^−/− cell by application of 1 μm TG 5 min prior to seal formation. After recording traces in 0 and 20 mm ${\text{Ca}}_0^{2+}$ solutions, the cell was exposed to 50 μm 2-APB. Shown here are peak current amplitudes recorded during steps to −100 mV. B, responses to voltage ramps recorded in the absence or presence of 2-APB at the times shown in A. C, I_CRAC amplitude (at −100 mV, normalized to cell capacitance) in IP₃R^−/− mutant (−/−; n = 6) and wild-type (+/+; n = 5) DT40 cells. D, comparison of potentiation and inhibition of I_CRAC by 50 μm 2-APB in wild-type (n = 5) and IP₃R^−/− mutant (n = 6) DT40 cells.

Application of 50 μm 2-APB to the IP₃R^−/− mutant DT40 cells produced a transient elevation of I_CRAC followed by a slow inhibition (Fig. 11A). The magnitudes of both the peak potentiation and the steady-state inhibition caused by 2-APB were identical to those seen in the control DT40 and Jurkat T cells (Fig. 11D). In addition, 2-APB diminished the extent of fast inactivation over time, just as it does in control DT40 cells (data not shown). Thus, we conclude that the complex stimulatory and inhibitory effects of 2-APB on CRAC channels occur through a mechanism that is independent of IP₃ receptors.

DISCUSSION

In this study, we have described complex effects of 2-APB on the amplitude and kinetic behaviour of I_CRAC. These include an increase of current amplitude (potentiation) and speeding of inactivation by low concentrations of 2-APB (< 5 μm), as well as inhibition of the current and removal of fast inactivation by higher concentrations (≥ 5 μm). Importantly, none of these effects appeared to occur through the IP₃ receptor, as previously postulated by the conformational coupling hypothesis for store-operated Ca²⁺ entry. In the sections that follow, we discuss some possible underlying mechanisms and implications of these phenomena.

2-APB exerts complex effects on the amplitude and kinetics of I_CRAC

We were surprised to find that 2-APB had much more complex effects on I_CRAC than only the slow inhibition reported previously (Braun et al. 2001). Low doses (1–5 μm) reversibly enhanced the amplitude of I_CRAC by up to fivefold and slightly increased the rate of fast Ca²⁺-dependent inactivation. At higher doses (≥ 5 μm) of the drug, the slow inhibition of I_CRAC also became apparent, along with the disappearance of fast inactivation. All of these effects were seen in Jurkat, RBL and DT40 cells; thus, they appear to be independent of the cell type (T cells, mast cells and B cells) and species (human, rat and chicken), suggesting that they may apply to CRAC channels in general. Thus, the reason why potentiation was not observed in a previous study of RBL cells (Braun et al. 2001) probably stems not from the cell type but from a combination of other factors. The amplitude of potentiation was less pronounced in RBL cells than in Jurkat or DT40 cells (compare Fig. 8C with Fig. 8A and 3A), and this smaller effect may have been exacerbated in other studies by the use of high concentrations of 2-APB (100 μm), which produce a predominant inhibitory effect, and by pre-application of 2-APB rather than acute application after I_CRAC had been activated.

The degree to which these 2-APB effects are specific for CRAC channels is mostly unknown. The inhibitory effects of 2-APB seem to be relatively non-specific, as they apply to IP₃ receptors (Sugawara et al. 1997), endogenous store-operated channels in HEK 293 cells (Ma et al. 2000) and the mitochondrial Ca²⁺ release machinery (Fig. 1B) in addition to I_CRAC. However, to our knowledge, 2-APB is the first example of a pharmacological agent that can enhance the activation of I_CRAC beyond the level achieved by complete store depletion. Because Ca²⁺ entry through CRAC channels is a key requisite step for the activation of T cells by antigen, potentiation of I_CRAC may be useful as a novel therapeutic approach to boost activation of immune cells in immunodeficient individuals. In addition, potentiation by 2-APB may prove to be a useful diagnostic tool for testing putative CRAC channel genes and probing the physiological functions of CRAC channels in vivo.

Possible mechanisms for the potentiation of I_CRAC by 2-APB

In resting cells in which Ca²⁺ stores were full and which displayed no Ca²⁺ influx through CRAC channels, low doses of 2-APB were ineffective in raising [Ca²⁺]_i or activating I_CRAC. Thus, rather than activating I_CRAC by itself, 2-APB appears to boost the activity of CRAC channels once they are activated by store depletion (Fig. 3E). This potentiation does not result from an increased emptying of Ca²⁺ stores, since 2-APB increases I_CRAC severalfold even after treatment with TG, which is known to deplete the ionomycin-releasable stores in Jurkat cells by > 90 % (see Fig. 1A of Hoth et al. 1997).

Although 2-APB does reduce the level of Ca²⁺-dependent fast inactivation at doses as low as 5 μm, this effect is not responsible for potentiation of the current. Conditions that limit or prevent fast inactivation, including holding potentials more positive than +30 mV or the use of Ba²⁺ as the charge carrier, did not reduce the amount of potentiation by 2-APB (Fig. 7). In principle, 2-APB could act by interfering with other known forms of I_CRAC inactivation, such as slow Ca²⁺-dependent inactivation (Zweifach & Lewis, 1995a) or kinase-dependent inactivation (Parekh & Penner, 1995). However, this seems unlikely, as both processes would be minimized by our recording conditions (10 mm EGTA and 0 ATP in the recording pipette).

In general, potentiation of I_CRAC could occur through increases in the number of activatable channels (N), the single-channel current (i) or the open probability of the channels (P_o). Without single-channel recordings, it is difficult to discriminate between these possible mechanisms, but we can make some educated guesses by exploiting the changes in fast inactivation that accompany I_CRAC potentiation. Previous work has shown that I_CRAC inactivation is influenced by the local [Ca²⁺]_i around individual CRAC channels (Zweifach & Lewis, 1995b). 2-APB at low concentrations does not induce inactivation by itself, since Ba²⁺ currents do not inactivate even in the presence of the drug (Fig. 7C); instead, a reasonable conclusion is that it increases the speed of fast Ca²⁺-dependent inactivation, possibly by producing an increase in the local [Ca²⁺]_i around CRAC channels. Fast inactivation is strongly dependent on the amplitude of i but not on N (Zweifach & Lewis, 1995b). Thus, if potentiation were due to an increase in i, then one would expect a significant increase in the extent of inactivation, particularly under conditions where the current increases significantly (e.g. by a factor > 2; see Fig. 5). This was not observed, suggesting that i is probably not affected by 2-APB. In addition, because CRAC channels inactivate independently (Zweifach & Lewis, 1995b), an increase in N would not be expected to affect inactivation. It might be argued that overlap of Ca²⁺ microdomains between adjacent CRAC channels might increase with N, undermining channel independence and increasing inactivation. To test this possibility, we reduced the current with SKF 96365, reasoning that this would reduce the overlap of microdomains and therefore reduce the inactivation if overlap existed. We found that, in the presence of a potentiating dose of 2-APB, a half-blocking dose of SKF 96365 did not affect the rate of inactivation (data not shown), making the overlap of domains unlikely.

If 2-APB does not influence N or i, the most likely explanation is that 2-APB potentiates I_CRAC by increasing P_o. The resulting increase in accumulation of intracellular Ca²⁺ at the inactivation sites would be expected to enhance inactivation, though perhaps to a smaller degree than would be expected from a comparable increase in i. A mechanism of this sort has been proposed to underlie the speeding of inactivation of voltage-gated L-type Ca²⁺ channels by Bay K 8644, which increases the P_o of these channels (Noceti et al. 1998). Further experiments on I_CRAC at the single-channel level will be needed to resolve this issue.

Inhibition of I_CRAC and the removal of inactivation by 2-APB

At concentrations ≥ 5 μm, 2-APB slowly inhibited I_CRAC and removed fast inactivation. The reversal of these effects after 2-APB was washed out of the bath was even slower and was often incomplete. In this sense, the inhibition by 2-APB is similar to that by imidazole compounds like SKF 96365 or econazole (Franzius et al. 1994; Christian et al. 1996). It is not known whether 2-APB (or SKF 96365) acts by blocking the channel pore directly, or by altering channel gating. Our data suggest that 2-APB does not act through a simple 1:1 interaction with the CRAC channel pore. The on-rate for inhibition, calculated from the time constant of I_CRAC inhibition in Fig. 6B, and K_1/2 for current inhibition (10 μm, Fig. 4B) was 1/500 μm⁻¹ s⁻¹. Thus, a simple 1:1 binding interaction would predict an off-rate of ∼1/50 s⁻¹, or a time constant of recovery of ∼50 s. This is much faster than the observed recovery time (22 % recovery at 160 s; estimated recovery time constant of > 600 s). The discrepancy cannot be explained by slow washout of the drug, as the potentiation effect of 2-APB reversed rapidly after drug removal (Fig. 3A). Instead, these results suggest that the mechanism of inhibition is more complex than simple pore blockade, and that the recovery from inhibition is not limited by the dissociation rate of 2-APB from its target.

2-APB inhibited both the amplitude of I_CRAC and the extent of fast inactivation with similar time courses, raising the intriguing possibility that these two effects may be causally linked. It is possible, for example, that 2-APB at high concentrations interrupts the coupling between the CRAC channel and a component that confers fast inactivation, and the activity of the CRAC channel declines because this component is necessary for maintaining the channel in an activatable state. Such a mechanism might also explain the kinetic discrepancy discussed above, if recovery of the inactivation component were much slower than the unbinding of 2-APB. Further experiments on the molecular underpinnings of fast inactivation may give clues as to the mechanism of I_CRAC inhibition by 2-APB.

IP₃ receptors are not needed for I_CRAC activation or inactivation

The mechanism underlying the activation of store-operated Ca²⁺ channels has been an area of active debate and, despite considerable efforts, no consistent picture has emerged. The leading candidate hypotheses include activation by a diffusible factor released from depleted stores, fusion of store-operated channel-laden vesicles with the plasma membrane, and direct activation of store-operated channels by conformational coupling to IP₃ receptors in the ER membrane (for reviews, see Putney & McKay, 1999; Prakriya & Lewis, 2001a). The conformational coupling hypothesis has received much recent support from evidence that IP₃ receptors bind directly to some members of the TRP family, thought by some to be the molecular correlates of CRAC channels (Kiselyov et al. 1998, 1999a; Boulay et al. 1999). Moreover, inhibitors of IP₃ receptors, such as 2-APB and heparin, also inhibit store-operated Ca²⁺ entry and the currents mediated by the putative store-operated channel TRP3 (Kiselyov et al. 1998; Ma et al. 2000). It has been suggested that fast inactivation of CRAC channels may arise through the inhibitory effects of Ca²⁺ on IP₃ receptors to which they are coupled (Berridge, 1995).

Our data are inconsistent with a necessary role for IP₃ receptors in the activation and inactivation of I_CRAC. First, we found that heparin failed to affect activation or the maintenance of I_CRAC in store-depleted cells, even at a concentration that completely prevented the ability of IP₃ to activate I_CRAC. A similar result was described recently in RBL cells (Broad et al. 2001). In contrast, currents mediated by recombinant TRP3 channels in excised patches from HEK 293 cells or endogenous ‘CRAC-like’ channels from A431 cells require addition of IP₃ and are blocked potently by heparin (Kiselyov et al. 1998, 1999b). Thus, the ability to activate I_CRAC through passive depletion, coupled with the inability of heparin to prevent this, suggests that CRAC channels differ from these other channels in not requiring even background levels of IP₃ for activation. Second, the activation and properties of I_CRAC were apparently normal in DT40 cells in which all three subtypes of IP₃ receptor had been knocked out by homologous recombination. These results confirm and extend earlier work by Sugawara et al. (1997) showing that TG was able to induce Ca²⁺ entry in these cells. Thus, our results show that CRAC channels do not require IP₃ receptors or IP₃ for their activation. However, these results do not rule out conformational coupling involving other proteins.

2-APB affects CRAC channel gating independently of IP₃ receptors

In the mutant DT40 cells lacking all three subtypes of IP₃ receptors, 2-APB elicited the same combination of I_CRAC potentiation, inhibition and changes in fast inactivation as in wild-type cells. Thus, the actions of 2-APB on I_CRAC are probably unrelated to any effect on IP₃ receptors. These results therefore show that inhibition of Ca²⁺ entry by 2-APB cannot be used to test the conformational coupling model for I_CRAC activation.

If IP₃ receptors are not involved, what then is the target for 2-APB's effects on CRAC channel activity? One possible site of action we considered was mitochondria, as 2-APB inhibited Ca²⁺ release from these organelles (Fig. 1B). In Jurkat T cells, uptake and release of Ca²⁺ by mitochondria is required to enable the cells to maintain a high rate of Ca²⁺ entry through CRAC channels (Hoth et al. 1997). To test the possibility that the effects of 2-APB on I_CRAC are an indirect consequence of modulating mitochondrial function, we treated cells with 1 μm antimycin A1 + 1 μm oligomycin to inhibit mitochondrial Ca²⁺ uptake. The inhibitors did not alter the ability of 2-APB to potentiate and inhibit I_CRAC (data not shown). Therefore, the effects of 2-APB on I_CRAC appear to be distinct from its effects on mitochondrial Ca²⁺ transport.

2-APB was more effective at modulating I_CRAC when applied extracellularly than when it was applied intracellularly through the recording pipette. A similar result was reported for RBL cells by Braun et al. (2001), who concluded that the drug's site of action was extracellular. The interpretation of this experiment is complicated by several factors. First, 2-APB may exist in multiple forms, including a protonated and unprotonated monomer, as well as a dimer (van Rossum et al. 2000), but the relative efficacy of these various compounds in affecting Ca²⁺ entry in general is unknown. Second, the ability of lipophilic compounds to interact with intracellular sites when applied intracellularly via a patch pipette is a function of their membrane permeability. Thus, when applied through the recording pipette, the concentration of a hydrophobic form of 2-APB near the plasma membrane may be much lower than expected, due to the fact that the rate-limiting diffusion step for such a hydrophobic compound may be across the tip of the pipette rather than across the plasma membrane. We reasoned that increasing the protonation of the drug should reduce permeability to some degree, and this should reduce the efficacy of extracellular drug if its site of action is intracellular. This approach has been applied previously in studies of membrane-permeant K⁺ channel blockers. For example, methadone is known to block K⁺ channels at an intracellular site, yet it is effective only when applied from outside the cell. Acidification of the extracellular medium was shown to inhibit the blocking action of methadone by reducing the concentration of the uncharged, lipophilic species (Horrigan & Gilly, 1996). In our experiments, reducing extracellular pH from 7.4 to 6.4 failed to decrease either potentiation or inhibition by 2-APB, implying that lowering the concentration of the unprotonated (and presumably membrane-permeant) form of 2-APB had no effect. The relative amounts of the protonated and unprotonated forms of 2-APB will depend on its pK_a, which is unknown, but is likely to be determined by its ethylamine group (pK_a= 10.8; Weast & Lide, 1989). Based on this pK_a value, one would predict that only 0.04 % of the 2-APB will be unprotonated at pH 7.4, but that this fraction will decrease by a factor of 10 at pH 6.4. Thus, the lack of an effect from lowering the pH (Fig. 10C) is consistent with an extracellular but not an intracellular site of action. However, given the various complications outlined above, a direct examination of this issue using membrane-impermeant variants of 2-APB will be needed to identify firmly the active species of 2-APB and its site of action.

The opposing effects of 2-APB on I_CRAC are reminiscent of the effects of the plant alkaloid ryanodine on the sarcoplasmic reticulum Ca²⁺ release channel in cardiac muscle. At submicromolar (1–50 nm) concentrations, ryanodine stabilizes the Ca²⁺ release channel in a subconductance state with a dramatically higher P_o, whereas at micromolar (10–1000 μm) concentrations, ryanodine closes the channel (Buck et al. 1992; Humerickhouse et al. 1993). These effects are manifested as increases in Ca²⁺ flux and muscle contraction by dilute ryanodine and inhibition of Ca²⁺ flux and muscle contraction by concentrated ryanodine. Binding studies indicate that the Ca²⁺ release channel has a high-affinity site, thought to mediate the stimulatory effects of ryanodine, and one or more low-affinity sites that mediate channel closure (Sutko et al. 1997). Thus, by analogy, it is possible that 2-APB has two separate binding sites, a high-affinity site that mediates I_CRAC potentiation and a low-affinity site that produces inhibition. Alternatively, different species of 2-APB may be responsible for the different effects: monomers, which are more prevalent at low concentrations, may mediate potentiation, whilst dimers, which are favoured at higher concentrations, may be responsible for inhibition. Further studies will be needed to resolve these issues.

In summary, we found that 2-APB elicits complex effects on I_CRAC. Low concentrations of 2-APB reversibly enhanced I_CRAC, whereas high concentrations also inhibited I_CRAC. 2-APB also altered the gating of I_CRAC by removing fast Ca²⁺-dependent inactivation. It was surprising to find that so many different properties of CRAC channels were affected, and an important question is whether the various effects of 2-APB are linked in some way or whether they are truly independent. Given that 2-APB affects the function of so many Ca²⁺ signalling proteins (IP₃ receptors, CRAC channels, TRP channels and the mitochondrial Ca²⁺ release mechanism), the drug clearly has limited utility as a specific inhibitor of store-operated Ca²⁺ entry. However, some effects of 2-APB (e.g. potentiation and removal of inactivation) may be sufficiently unique that identification of the parts of the molecule that are responsible for these effects and the proteins with which they interact could provide new clues to the mechanisms underlying activation, deactivation and inactivation of CRAC channels.