Abstract
3,5-Bis(trifluoromethyl)pyrazole derivative (BTP2) or N-[4-3, 5-bis(trifluromethyl)pyrazol-1-yl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (YM-58483) is an immunosuppressive compound that potently inhibits both Ca2+ influx and interleukin-2 (IL-2) production in lymphocytes. We report here that BTP2 dosedependently enhances transient receptor potential melastatin 4 (TRPM4), a Ca2+-activated nonselective (CAN) cation channel that decreases Ca2+ influx by depolarizing lymphocytes. The effect of BTP2 on TRPM4 occurs at low nanomolar concentrations and is highly specific, because other ion channels in T lymphocytes are not significantly affected, and the major Ca2+ influx pathway in lymphocytes, ICRAC, is blocked only at 100-fold higher concentrations. The efficacy of BTP2 in blocking IL-2 production is reduced approximately 100-fold when preventing TRPM4-mediated membrane depolarization, suggesting that the BTP2-mediated facilitation of TRPM4 channels represents the major mechanism for its immunosuppressive effect. Our results demonstrate that TRPM4 channels represent a previously unrecognized key element in lymphocyte Ca2+ signaling and that their facilitation by BTP2 supports cell membrane depolarization, which reduces the driving force for Ca2+ entry and ultimately causes the potent suppression of cytokine release.
In many cells, receptor activation evokes an increase in cytosolic Ca2+ concentration ([Ca2+]i) composed of release from intracellular stores followed by store-operated Ca2+ entry via calcium release-activated calcium (CRAC) channels (Penner et al., 1993; Parekh and Penner, 1997; Putney et al., 2001). These channels are functionally best characterized in mast cells (Hoth and Penner, 1992, 1993) and T lymphocytes (Zweifach and Lewis, 1993; Premack et al., 1994) and are considered key elements for the activation of immune cells. The study of CRAC has been hampered by the lack of potent and specific pharmacological tools. At present, the most widely used organic blockers of store-operated calcium entry pathways are imidazole antimycotics, such as econazole, the closely related compound SKF-96365, and 2-aminoethoxydiphenyl borate (Mason et al., 1993; Chung et al., 1994; Franzius et al., 1994; Christian et al., 1996; Hermosura et al., 2002). However, none of these compounds is very potent or selective.
A class of pyrazole derivatives, bis(trifluoromethyl)pyrazoles (BTPs), has been reported to act as potent immunosuppressive compounds by inhibiting cytokine release (IL-2, IL-4, IL-5, interferon-γ, and others) from human lymphocytes and suppressing T-cell proliferation (Djuric et al., 2000; Trevillyan et al., 2001; Chen et al., 2002; Ishikawa et al., 2003). In addition, these compounds have proven effective in various immune disease-relevant rodent and nonhuman primate models, in which they inhibit trinitrochlorobenzene-induced contact hypersensitivity in mice (a model of T lymphocyte-mediated delayed type hypersensitivity) and Ascaris suum-induced immediate bronchoconstriction of cynomolgus monkeys (an asthma model) (Djuric et al., 2000; Ishikawa et al., 2003). Despite the rather detailed characterization of the compound's potent effects in many immune-based cellular and animal models, the mechanism by which BTPs inhibit cytokine production in lymphocytes remains unknown. The effects of BTPs are presumably linked to intracellular Ca2+ signaling, because the pyrazole derivative BTP2 (Fig. 2D) potently inhibits thapsigargin-evoked Ca2+ influx in the low nanomolar range (Ishikawa et al., 2003), and two recent reports suggest that BTP2 may inhibit the store-operated Ca2+ current ICRAC (Zitt et al., 2004; He et al., 2005) and TRPC3 and TRPC5 channels (He et al., 2005).
The transient receptor potential (TRP) proteins represent an important family of mammalian ion channels that are believed to be involved in Ca2+ signaling. Based on sequence similarities, the mammalian TRP channel family is divided into three subfamilies: TRPC, TRPV, and TRPM (Harteneck et al., 2000; Clapham et al., 2001; Montell et al., 2002a,b). TRPM4, specifically the longer splice variant TRPM4b (which we will refer to as TRPM4 throughout this manuscript), is a widely expressed Ca2+-activated nonselective cation channel of the TRPM ion channel subfamily that does not conduct Ca2+ but instead mediates cell membrane depolarization (Launay et al., 2002). In electrically nonexcitable cells, a depolarization would tend to decrease the driving force for Ca2+ influx through store-operated Ca2+ channels. Many other ion channels, such as voltagedependent potassium (Kv1.3) channels (Lin et al., 1993; Hanson et al., 1999), intermediate/small-conductance calcium-activated potassium (IK/SK2) channels (Lin et al., 1993; Jensen et al., 1999; Desai et al., 2000; Wulff et al., 2000), and swelling-activated chloride (Clvol) channels (Lewis et al., 1993; Ross et al., 1994), are also known to be involved in the regulation of the driving force for Ca2+ influx, but they do so by hyperpolarizing the membrane potential (Penner et al., 1988; Cahalan and Chandy, 1997; Cahalan et al., 2001). Thus, the net Ca2+ influx into a cell is not only determined by Ca2+ entry pathways themselves, but also by ion channels that modulate the membrane potential.
In the present study, we investigated the mechanism by which BTP2 inhibits Ca2+ signaling in Jurkat T cells. We demonstrate that BTP2 potently and selectively facilitates the activity of TRPM4, resulting in reduced Ca2+ entry and cytokine release. This compound therefore represents a novel and promising pharmacological tool to inhibit Ca2+ signaling in lymphocytes and other cell types that regulate Ca2+ influx by the concerted actions of store-operated Ca2+ channels and Ca2+-activated cation channels.
Materials and Methods
Cells. HEK-293 cells transfected with the FLAG-human TRPM4b/pCDNA4/TO construct were grown on glass coverslips with Dulbecco's modified Eagle's medium supplemented with 10% FBS, blasticidin (5 μg/ml), and zeocin (0.4 mg/ml). TRPM4b expression was induced 1 day before use by adding 1 μg/ml tetracycline to the culture medium, and patch-clamp experiments were performed 16 to 24 h after induction (for details, see Launay et al., 2002). Human leukemic T cells (Jurkat cells) were grown in RPMI 1640 medium (10% FBS) and rat basophilic leukemia cells (RBL-2H3 cells) in Dulbecco's modified Eagle's medium (10% FBS).
Materials. BTP2 (or YM-58483) and SKF-96365 were synthesized by Astellas Pharma Inc. (Tokyo, Japan). Econazole, margatoxin, apamin, charybdotoxin, and stilbenedisulfonate 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid were purchased from Sigma and dissolved in dimethyl sulfoxide. Phytohemagglutinin (PHA) was obtained from Sigma (St. Louis, MO) and dissolved in phosphatebuffered saline.
Solutions. For ICRAC measurements, the standard bath solution had the following composition: 140 mM NaCl, 2.8 mM KCl, 10 mM CaCl2, 2 mM MgCl2, 10 mM CsCl, 10 mM glucose, and 10 mM HEPES·NaOH, pH 7.2, with osmolarity adjusted to approximately 320 mOsM. Intracellular pipette-filling solutions contained 140 mM cesium glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM cesium-BAPTA, and 10 mM HEPES·CsOH, pH 7.2, adjusted with CsOH. In experiments in which [Ca2+]i was buffered to elevated levels, CaCl2 was added as necessary [calculated with WebMaxC (http://www.stanford.edu/~cpatton/webmaxcS.htm), temperature = 24°C, pH = 7.2, ionic equivalent = 0.16]. Solution changes were performed by pressure ejection from a wide-tipped pipette.
Electrophysiology. Patch-clamp experiments were performed in the tight-seal whole-cell configuration at 21 to 25°C. High-resolution current recordings were acquired by a computer-based patch-clamp amplifier system (EPC-9; HEKA, Lambrecht, Germany). Patch pipettes had resistances between 2 and 4 MΩ after filling with the standard intracellular solution. Immediately after establishment of the whole-cell configuration, voltage ramps of 50- to 200-ms duration spanning the voltage range of -100 to +100 mV were delivered at a rate of 0.5 Hz over a period of 300 to 400 s. All voltages were corrected for a liquid junction potential of 10 mV between external and internal solutions when using glutamate as an intracellular anion. Currents were filtered at 2.9 kHz and were digitized at 100-μs intervals. Capacitive currents and series resistance were determined and corrected before each voltage ramp using the automatic capacitance compensation of the EPC-9. The low-resolution temporal development of membrane currents was assessed by extracting the current amplitude at -80 or +80 mV from individual ramp current records. Where applicable, statistical errors of averaged data are given as means ± S.E.M. with n determinations.
Voltage-dependent potassium (Kv1.3) currents were measured in Jurkat cells. The bath solution contained 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES·NaOH, pH 7.2. The internal solution contained 140 mM potassium glutamate, 2 mM MgCl2, 1 mM CaCl2, 10 mM potassium-EGTA, and 10 mM HEPES·KOH, pH 7.2. Ramps were given every 30 s (-100 to +100 mV in 200 ms), and cells were held at -80 mV between ramps. Currents were not leak-subtracted.
Calcium-activated potassium (SK2) currents were measured in Jurkat cells. The bath solution contained 164.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES·KOH, pH 7.2. The internal solution contained 135 mM potassium aspartate, 2 mM MgCl2, 1.1 mM potassium EGTA, and 10 mM HEPES·KOH, pH 7.2. Ramps were given every2s(-100 to +40 mV in 200 ms), and cells were held at -80 mV between ramps. Free intracellular [Ca2+]i was adjusted to 1 μM.
Swelling-activated chloride (Clvol) currents were measured in Jurkat cells. The bath solution contained 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 5 mM HEPES·NaOH, pH 7.2. The internal solution contained 160 mM cesium glutamate, 2 mM MgCl2, 0.1 mM CaCl2, 1.1 mM cesium-EGTA, 4 mM sodium ATP, 100 mM glucose, and 10 mM HEPES·CsOH, pH 7.2. Ramps were given every 2 s (-100 to +50 mV in 200 ms), and cells were held at -60 mV between ramps.
TRPM4 currents were measured in Jurkat and HEK-293 cells overexpressing TRPM4b. The bath solution contained 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM HEPES·NaOH, pH 7.2. The internal solution contained 120 mM potassium glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM potassium-BAPTA, and 10 mM HEPES·KOH, pH 7.2. Ramps were given every 2 s (-100 to +100 mV in 50 ms), and cells were held at -80 mV between ramps. Free intracellular [Ca2+]i was adjusted as indicated in the figure legends.
IL-2 Production Assay. Jurkat cells (5 × 106 cells/ml) were placed in a 96-well microplate and incubated with either 10 μg/ml or 2 μg/ml PHA (Sigma) for 24 h, and the supernatant was collected from these cells after centrifugation (200g, 24°C for 3 min). The concentration of IL-2 was measured by the human IL-2 ELISA system (human IL-2 ELISA Kit DuoSet; Genzyme Co., Cambridge, MA). Optical density values at 450 nm were measured with the use of a microplate reader (Spectra Max 190; Molecular Devices, Sunnyvale, CA). Data for BTP2-treated cells were normalized to those of untreated control cells.
Calcium Measurements. For Ca2+ measurements, Fura-2 acetoxymethyl ester-loaded cells (5 μM/30 min at 37°C) were kept in standard extracellular saline containing 140 mM NaCl, 2.8 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES·NaOH, pH 7.2, and excited by wavelengths of 340 and 380 nm. Fluorescence emission of several cells was simultaneously recorded at a frequency of 1 Hz using a dual excitation fluorometric imaging system (TILL-Photonics, Gräfelfingen, Germany) controlled by TILL-Vision software. Signals were computed into relative ratio units of the fluorescence intensity of the different wavelengths (340/380 nm).
Results
Effect of BTP2 on ICRAC. Because BTP2 inhibits thapsigargin-induced Ca2+ influx (Ishikawa et al., 2003) and reportedly inhibits the store-operated calcium current ICRAC (Zitt et al., 2004), we analyzed the effects of BTP2 on ICRAC in RBL-2H3 and Jurkat T cells. ICRAC was activated by a standard protocol after whole-cell break-in with 20 μM InsP3 in the pipette solution. The time course of whole-cell current was monitored by 200-ms voltage ramps that spanned -100 to +100 mV and were applied every 2 s. Each voltage ramp yields a full current-voltage (I-V) relationship (Fig. 1C), and the ICRAC time course is shown in Fig. 1A. It was constructed by measuring the whole-cell current amplitude on the ramp trace corresponding to -80 mV, normalizing the current amplitude to the value obtained just before the application of compound, and plotting it as a function of time. Extracellularly applied BTP2 blocked InsP3-evoked ICRAC in Jurkat cells in a concentration-dependent manner (Fig. 1A). Complete inhibition of the current was obtained within 200 s after the addition of 10 μM BTP2. Lower concentrations also blocked, but they did so more slowly. Figure 1E shows the dose-response relationship for BTP2-mediated block of ICRAC 300 s after application, along with the dose-response curve obtained for the reference compound econazole under identical experimental conditions (raw data not shown). In Jurkat cells, the apparent IC50 values for BTP2 and econazole were 2.2 and 4.0 μM, respectively (Hill coefficient = 1.7 each).
Next, we studied the effect of BTP2 on ICRAC in RBL-2H3 cells and observed that BTP2 also blocks InsP3-evoked ICRAC in these cells in a concentration-dependent manner (Fig. 1B). With an IC50 value of 0.5 μM (Fig. 1F; Hill coefficient = 1.4), BTP2 seems slightly more potent in RBL cells than in Jurkat cells, whereas the potency of econazole in RBL cells (IC50 = 4.2 μM; Hill coefficient = 1.4) was similar to that observed in Jurkat cells. To study the site of action, we included BTP2 in the pipette solution. Intracellularly applied BTP2 (10 μM) did not exhibit any inhibitory effect on ICRAC in either RBL-2H3 cells or Jurkat cells, and it did not affect the block of ICRAC induced by extracellularly applied BTP2 in either cell type (Fig. 1D). This suggests that the inhibitory action of BTP2 on ICRAC may be located extracellularly; however, we cannot completely rule out the possibility that the lipophilicity of the compound might allow it to escape rapidly across the plasma membrane before affecting the channel.
Although the above results demonstrate that BTP2 can inhibit ICRAC in both RBL and Jurkat cells, the potency seems to be too low to account for the compound's ability to suppress cytokine production, which occurs in the low nanomolar range. Zitt et al. (2004) reported recently that ICRAC is inhibited by BTP2 at lower concentrations, but this effect requires hours to develop, and they arrived at an IC50 value of approximately 10 nM after 24 h of preincubation with the compound (Zitt et al., 2004). He et al. (2005) reported that store-operated Ca2+ entry was reduced much quicker, within approximately 10 min, but the concentrations required to do so in HEK-293 cells and DT40 B cells were found to be at least 1 order of magnitude higher (He et al., 2005), similar to the efficacy of BTP2 in blocking ICRAC in RBL cells (Fig. 1F). To further assess the involvement of ICRAC, we analyzed the kinetics of BTP2 action on both Ca2+ signaling and cytokine production. Figure 1G illustrates the effect of 100 nM BTP2 on Jurkat Ca2+ signals evoked by 10 μg/ml PHA. This relatively low concentration of BTP2 suppressed the PHA-induced Ca2+ signals almost completely within approximately 10 min and therefore cannot be reconciled easily with the reported slow ICRAC inhibition (time constant = 98 min) (Zitt et al., 2004). We next analyzed the temporal development of the efficacy of BTP2 to suppress IL-2 release over variable time spans ranging from 2 to 24 h. Jurkat cells were stimulated with 10 μg/ml PHA, and IL-2 production was measured by ELISA at various times after the coapplication of PHA and various concentrations of BTP2. Figure 1H illustrates that there was no significant change in the potency with which BTP2 suppressed IL-2 release, because the IC50 value remained constant in the low nanomolar range even when measuring IL-2 release as early as 2 h after stimulation, again suggesting that the main mechanism of action of BTP2 is engaged quickly and does not require several hours to develop. This would suggest that the ICRAC inhibition observed by Zitt et al. (2004) at low nanomolar concentrations may be caused by another event rather than as a direct effect on CRAC channels. Because BTP2 has been demonstrated to suppress lymphocyte proliferation (Trevillyan et al., 2001; Zitt et al., 2004), one possible reason for the decrease in ICRAC amplitude after long BTP2 preincubations could be that lymphocytes become arrested at a certain cell-cycle stage in which ICRAC is suppressed. Indeed, serum-starved RBL cells become arrested in G0/G1 and have been reported to lack significant ICRAC (Bodding, 2001). He et al. (2005) found a relatively fast inhibition of store-operated Ca2+ influx, but the concentrations required probably reflect the inhibition of ICRAC we observe in, for example, RBL cells. Given that the effect of BTP2 even at low concentrations is relatively quick compared with the time course over which it compromises ICRAC, we considered alternative or additional mechanisms that BTP2 may be targeting.
Effect of BTP2 on Other Ionic Currents in Jurkat Cells. Whole-cell recordings permit the direct measurement of several types of channel activity that may affect Ca2+ signaling in Jurkat cells such as the voltage-dependent potassium channel Kv1.3, the small-conductance Ca2+-activated potassium channel SK2, and volume regulatory Cl- channel Clvol. We first analyzed the efficacy of known inhibitors of these channels on PHA-stimulated cytokine production in Jurkat T lymphocytes. Of the presumed ICRAC inhibitors, BTP2, econazole, and SKF-96365 (10 μM each), only BTP2 completely inhibited PHA-induced IL-2 production (Fig. 2A), consistent with its complete inhibition of ICRAC at that concentration. Econazole also suppressed cytokine production, albeit not completely, whereas SKF-96365 was not very effective. The other inhibitors exhibited varying degrees of inhibition. The K+ channel blockers, including the Kv1.3-specific inhibitor margatoxin (1 nM), the SK2 inhibitor apamin (10 nM), and the IK inhibitor charybdotoxin (100 nM), produced inhibitory effects in the range of 30 to 50%, whereas the Clvol channel inhibitor stilbenedisulfonate 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid (100 μM) was ineffective. The efficacy of K+ channel blockers to affect IL-2 production is consistent with the fact that they inhibit ion currents which promote Ca2+ influx via a membrane hyperpolarization. However, none of the compounds was able to suppress cytokine production completely. Moreover, none of the K+ and Cl- channel inhibitors tested in this study exhibited significant additive inhibitory effects on IL-2 production when it was already partially inhibited by 10 nM BTP2 (Fig. 2B).
To assess the specificity of BTP2, we tested for its effects on the K+ and Cl- channels expressed in Jurkat cells. BTP2 did not significantly affect the activity of any of these channels, even at the highest concentration (10 μM) tested (Fig. 2C), with the possible exception of Cl- currents, which were inhibited by ∼50%. In addition, BTP2 showed no inhibitory effect on ADP-ribose-activated TRPM2 channels expressed in HEK-293 cells (Perraud et al., 2001; Sano et al., 2001) and inward rectifier K+ currents in RBL cells (Lindau and Fernandez, 1986). Given the significant discrepancy in the potency of BTP2 to inhibit IL-2 production at low nanomolar concentrations versus ICRAC inhibition with an IC50 value of 2.1 μM and the lack of effect on K+ channels, we considered TRPM4, a CAN channel expressed in Jurkat cells (Launay et al., 2002, 2004), to be a possible target.
Voltage and [Ca2+]i Dependence of TRPM4. We have shown previously that TRPM4 can be detected at RNA and protein levels in various T cells of murine and human origin and that these channels are functional in Jurkat T cells (Launay et al., 2004). Moreover, we have observed calciumactivated TRPM4-like currents in human primary T cells (A. Beck and R. Penner, unpublished observations). To assess TRPM4 function, we used Jurkat T cells as a model system and carried out experiments very similar to those described in Fig. 1A, except that the free Ca2+ concentration of the intracellular pipette solution was buffered to levels of 0.3 to 1.8 μM. Under these conditions, Jurkat cells indeed produce large cation currents with current-voltage signatures indistinguishable from those of TRPM4, and we refer to these currents as ICAN. In initial experiments, application of BTP2 to maximally activated ICAN did not yield any significant modification of the currents, either inhibitory or facilitatory (data not shown). However, these experiments were carried out at a holding potential of 0 mV, which yields maximal activation of ICAN in Jurkat cells (Fig. 4A), whereas the resting potential of these cells is considerably more negative. The resting membrane potential is an important factor in determining TRPM4 behavior, because the channel exhibits a striking voltage dependence (Launay et al., 2002; Hofmann et al., 2003; Nilius et al., 2003). To assess the influence of membrane voltage on TRPM4 activity, we studied the voltage dependence of TRPM4 in HEK-293 cells.
Figure 3A illustrates the activation of Ca2+-activated currents in TRPM4 overexpressing HEK-293 cells kept at various membrane holding potentials and perfused with a solution in which [Ca2+]i was buffered to 1.3 μM. Exemplary current-voltage relationships of TRPM4 obtained by voltage ramps spanning -100 to +100 mV and delivered from holding potentials of -60 and +60 mV are illustrated in Fig. 3B. They illustrate the typical characteristics of TRPM4 as reported previously (Launay et al., 2002; Hofmann et al., 2003; Nilius et al., 2003). The data sets obtained at different holding potentials demonstrate that the magnitude of inward currents carried by TRPM4 is strongly dependent on the holding potential from which the voltage ramps are delivered. The maximum current amplitude obtained when holding the cell membrane potential at negative values is considerably smaller than that obtained at more depolarized holding potentials (Fig. 3A). The bar graph of Fig. 3C illustrates the voltage dependence of maximum inward currents obtained by perfusing cells with 1.8 μM free Ca2+.
A more comprehensive analysis of this voltage dependence is presented in Fig. 3D, in which we plot the maximum current amplitudes of TRPM4 currents (extracted from ramp currents at -80 mV) as a function of [Ca2+]i and measured in cells that were held at different holding potentials (-60, -30, and +60 mV). From this analysis, it is clear that the degree of TRPM4 activation is dependent on both [Ca2+]i and the membrane potential, with increasing Ca2+ and positive voltages synergizing to recruit larger maximal currents. The dose-response curves fitted to the various data sets of Fig. 3D reveal that the holding potential does not affect the responsiveness of TRPM4 to [Ca2+]i, because the apparent EC50 for TRPM4 activation remains fairly constant at ∼500 nM, regardless of the holding potential (Fig. 3D). From this, we infer that the primary effect of membrane potential on TRPM4 activity resides at the level of the channel's open probability, which is consistent with our previous observations in single-channel recordings of TRPM4, in which negative membrane voltages strongly reduced their open probability (Launay et al., 2002).
Effect of BTP2 on TRPM4 in HEK-293 Cells. Based on the above observations, we reasoned that BTP2 could potentially increase the open probability of TRPM4 at negative membrane potentials. We therefore kept the holding potential of HEK-293 cells overexpressing TRPM4 at -80 mV and then tested BTP2 for possible augmentation of inward currents. Figure 3E illustrates the average inward currents carried by TRPM4 at -80 mV with [Ca2+]i clamped at 500 nM. Control cells indeed showed a significantly reduced TRPM4 current at this negative holding potential, and these were greatly enhanced when cells were pretreated with 10 μM BTP2 for several minutes. The current-voltage relationships illustrated in Fig. 3F were obtained from representative control and BTP2-treated cells at 170 s after whole-cell establishment, and they demonstrate the massive up-regulation of TRPM4 currents by BTP2. These results suggest that BTP2 can facilitate TRPM4 channels overexpressed in the heterologous expression system.
Effect of BTP2 on ICAN in Jurkat T Cells.Fig. 4A demonstrates that ICAN in Jurkat cells also exhibits strong voltage dependence. Cells kept at a holding potential of 0 mV and perfused with pipette solutions in which [Ca2+]i was buffered to 500 nM yielded large cation currents (Fig. 4A) with the typical current-voltage relationship of TRPM4 (Fig. 4B). Under the same experimental conditions, cells kept at a holding potential of -80 mV exhibited strongly reduced ICAN current amplitudes (Fig. 4A), quite analogous to the observations made with TRPM4 in the heterologous expression system (Fig. 3A). Under these conditions, BTP2 did not enhance any currents when [Ca2+]i was buffered to resting levels of 100 nM or lower (data not shown). However, when intracellular solutions were buffered to a slightly elevated [Ca2+]i level of 500 nM, the preincubation of Jurkat cells with various concentrations of BTP2 resulted in a dose-dependent enhancement of Ca2+-activated inward currents (Fig. 4C). The effect manifested itself in both an increase in maximal current amplitude and in the acceleration of the kinetics with which ICAN developed. We analyzed the facilitation of ICAN by constructing dose-response relationships for BTP2-mediated increases in inward currents as a function of BTP2 concentration 300 s after whole-cell recording (Fig. 4D). The apparent half-maximal effective concentration (EC50) was 8 nM. Thus, the potency of BTP2 to enhance ICAN is roughly 100-fold higher than that of inhibiting ICRAC and close to its efficacy in inhibiting IL-2 production. We therefore propose that the primary action of BTP2 is based on its ability to shift the voltage dependence of TRPM4, so that open probability is increased at more negative membrane potentials. This effect enhances ICAN-mediated cell membrane depolarization and thereby reduces the driving force for Ca2+ influx.
We sought to further corroborate the above hypothesis by assessing IL-2 production in Jurkat cells under experimental conditions that would negate the depolarizing action of ICAN. This can be accomplished by extracellular solutions in which Na+, the primary charge carrier of ICAN, is replaced by an impermeant cation such as choline. This largely prevents TRPM4-mediated membrane depolarization and has been shown to enhance Ca2+ influx (Launay et al., 2002). We analyzed the effect of BTP2 on PHA-stimulated IL-2 production in Jurkat cells cultured in the presence or absence of Na+ in the extracellular medium. As illustrated in Fig. 4E, BTP2 potently inhibited IL-2 production in the presence of Na+ with an IC50 value of 0.25 nM. This value is lower than the IC50 value reported previously of ∼10 nM and may be a consequence of the Ringer's-like extracellular solution used in the present study. When performing the IL-2 assay in choline-based Na+-free media under otherwise identical conditions, the IC50 value for BTP2-mediated inhibition of IL-2 production was reduced by approximately 100-fold (IC50 = 14 nM). This result is entirely consistent with the notion that at low concentrations, BTP2 acts primarily via the facilitation of ICAN. The specificity of the Na+ removal experiment is demonstrated by the dose-response curves of econazole-mediated inhibition of IL-2 production under the same experimental conditions (Fig. 4F). Here, the removal of Na+ did not alter the efficacy of econazole to suppress cytokine production.
Discussion
Our results establish a novel principle in regulating cytokine release from lymphocytes, a process that is intimately linked to elevations in [Ca2+]i. We demonstrate that the pyrazole compound BTP2, a potent inhibitor of IL-2 release in lymphocytes, effectively facilitates the activity of the [Ca2+]i-activated nonselective cation channel TRPM4 both in a heterologous expression system and in Jurkat cells, which express the protein natively. The efficacy of facilitating ICAN and the ability to suppress cytokine production occur in the low nanomolar concentration range within a few minutes of BTP2 exposure, suggesting that the primary mechanism of action of BTP2 is due to its effect on TRPM4. At higher concentrations, the compound can also inhibit ICRAC. Even at these higher concentrations, the specificity of BTP2 for ICRAC seems remarkable compared with the effects on a range of other ion channels found in lymphocytes. Thus, of all compounds known presently to inhibit ICRAC, BTP2 represents the most potent and most selective ICRAC inhibitor, even though its primary mechanism of action seems linked to TRPM4.
BTP2 seems to be a very potent inhibitor of T-cell activation in the low nanomolar range (Djuric et al., 2000; Trevillyan et al., 2001; Chen et al., 2002; Ishikawa et al., 2003); see Figs. 1 and 4), and it inhibits [Ca2+]i signals within a few minutes (Fig. 1G). Any mechanism that accounts for this effect should match the potency and kinetic aspects of BTP2 action. So far, three mechanisms of BTP2 have been proposed to account for its inhibitory effects on Ca2+ influx and cytokine production: the inhibition of the store-operated current ICRAC (Zitt et al., 2004; He et al., 2005), the inhibition of the TRPC channels TRPC3 and TRPC5b (He et al., 2005), and the facilitation of TRPM4 (the present study).
All three studies agree that store-operated Ca2+ influx is compromised by BTP2; however, discrepancies exist in terms of potency and kinetics of block. Zitt et al. (2004) attribute BTP2's potent suppression of Ca2+ influx in lymphocytes in the low nanomolar range to a slow block of ICRAC that develops over hours. He et al. (2005) did not measure ICRAC directly but found that thapsigargin and receptor-mediated Ca2+ signals were inhibited within approximately 10 min, but the potency was at least 1 order of magnitude higher (IC50 ∼0.1-0.3 μM). It should be noted that the IC50 values reported by Gill and colleagues (He et al., 2005) are not directly comparable with the values provided by Zitt et al. (2004) or our own study, because they represent only indirect measures of channel activity as determined from changes in [Ca2+]i and therefore probably overestimate the true potency at the channel level. Our study finds that ICRAC in RBL and Jurkat T cells can indeed be inhibited relatively quickly within a few minutes, and potencies range from 0.5 to 4 μM as derived from direct current inhibition profiles. Whereas the study of Zitt et al. (2004) is compatible with the potency range of BTP2 on inhibition of cytokine production, the slow kinetics of ICRAC inhibition reported by Zitt et al. (2004) is at odds with the relatively fast inhibition of Ca2+ influx (Fig. 1G). Conversely, the studies by He et al. (2005) and our own data are compatible with the kinetics of inhibition of store-operated currents, but neither study found a low nanomolar potency that would be required to explain the efficacy of BTP2 in terms of ICRAC inhibition alone. Likewise, the efficacy of BTP2 in blocking other channels such as heterologously expressed TRPC3 and TRPC5, which He et al. (2005) estimate to be ∼0.3 μM, does not approach the low BTP2 concentrations that suffice to mediate the inhibition of cytokine release in native T cells. These channels also would not seem to be responsible for BTP-mediated inhibition of cytokine release from lymphocytes, because the only Ca2+-permeable current elicited by either antigen or thapsigargin stimulation seems to be ICRAC, and no ion channels that fit the profile of TRPC3 or TRPC5 have so far been reported in the literature. From these considerations and based on the data present in this study, it seems that the only mechanism which fits the criteria for BTP2 effects in T cells in terms of potency and kinetics is provided by the facilitation of TRPM4. Not only are the potency and kinetic properties adequate, but these channels have been identified in T cells as important factors in determining the amount of Ca2+ influx (Launay et al., 2004). At higher concentrations, ICRAC inhibition may contribute to this inhibition. Likewise, at these higher concentrations and in cell types that express TRPC3/TRPC5, the BTP2 effects may also involve inhibition of TRPC channels.
Acknowledgments
We thank Mahealani K. Monteilh-Zoller and Carolyn E. Oki for expert technical assistance.
Footnotes
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This work was supported in part by the following grants from the National Institutes of Health: R01-AI50200, R01-GM63954, and R01-NS40927 (to R.P.); R01-GM65360 (to A.F.); and R01-AI46734 (to J.-P.K.). P.L. was supported by a fellowship from Human Frontier Science Program Organization.
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ABBREVIATIONS: CRAC, calcium release-activated calcium (channel); TRP, transient receptor potential; TRPM4, transient receptor potential melastatin 4; BTP, bis(trifluoromethyl)pyrazole; BTP2, 3,5-bis(trifluoromethyl)pyrazole derivative; YM-58483, N-[4-3,5-bis(trifluromethyl)pyrazol-1-yl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; FBS, fetal bovine serum; IL, interleukin; CAN, Ca2+-activated nonselective; HEK, human embryonic kidney; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; PHA, phytohemagglutinin; ELISA, enzyme-linked immunosorbent assay; InsP3, inositol trisphosphate; I-V, current/voltage; SKF-96365, 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride.
- Received November 24, 2005.
- Accepted January 10, 2006.
- The American Society for Pharmacology and Experimental Therapeutics