Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on I_CRAC-like calcium channels

Abstract

Activation of phospholipase C in nonexcitable cells causes the release of calcium (Ca²⁺) from intracellular stores and activation of Ca²⁺ influx by means of Ca²⁺ release-activated channels (I_CRAC) in the plasma membrane. The molecular identity and the mechanism of I_CRAC channel activation are poorly understood. Using the patch–clamp technique, here we describe the plasma membrane Ca²⁺ channels in human carcinoma A431 cells, which can be activated by extracellular UTP, by depletion of intracellular Ca²⁺ stores after exposure to the Ca²⁺-pump inhibitor thapsigargin, or by loading the cells with Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate. The observed channels display the same conductance and gating properties as previously described I_min channels, but have significantly lower conductance for monovalent cations than the I_CRAC channels. Thus, we concluded that the depletion-activated Ca²⁺ current in A431 cells is supported by I_CRAC-like (I_CRACL) channels, identical to I_min. We further demonstrated synergism in activation of I_CRACL Ca²⁺ channels by extracellular UTP and intracellular inositol (1,4,5)-triphosphate (IP₃), apparently because of reduction in phosphatidylinositol 4,5-bisphosphate (PIP₂) levels in the patch. Prolonged exposure of patches to thapsigargin renders I_CRACL Ca²⁺ channels unresponsive to IP₃ but still available to activation by the combined action of IP₃ and anti-PIP₂ antibody. Based on these data, we concluded that phospholipase C-mediated and store-operated Ca²⁺ influx pathways in A431 cells converge on the same I_CRACL Ca²⁺ channel, which can be modulated by PIP₂.

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca²⁺ from intracellular Ca²⁺ stores and activation of Ca²⁺ influx across the plasma membrane by means of capacitative Ca²⁺ entry or store-operated Ca²⁺ entry processes (1–3). These processes are mediated by plasma membrane Ca²⁺ channels termed “Ca²⁺ release activated channels” (I_CRAC) (4–7). The molecular identity of I_CRAC remains unclear, with mammalian trp channels (mTrp) usually considered the most likely candidate for the role of I_CRAC (1–3, 8, 9). When compared with I_CRAC, mTrp channels display relatively low selectivity for divalent cations, higher single channel conductance, and different kinetic and pharmacological properties. In experiments with a human carcinoma A431 cell line, we previously described plasma membrane Ca²⁺ channels (I_min) that are activated by application of uridine triphosphate and bradykinin to cell-attached patches or by application of inositol (1,4,5)-trisphosphate (IP₃) to excised inside-out (i/o) patches (10–12). IP₃-gated channels that share some common properties with I_min have been also observed in experiments with human T cells (13), rat macrophages (12), and endothelial cells (14, 15). Major functional properties of I_min channels, such as small conductance (1 pS for divalent cations), high selectivity for divalent cations (P_Ca/K > 1,000), inward rectification, and sensitivity to block by SKF95365 are similar to I_CRAC channels (12, 16). Thus, we previously suggested that I_min and I_CRAC may in fact be the same channels (17).

The mechanism of I_CRAC activation remains similarly controversial (1–3). When studied in a heterologous expression system, activation of mTrp channels by IP₃ appear to be mediated by direct conformational coupling between the cytosolic carboxyl-terminal tail of mTrp and the amino-terminal ligand-binding domain of intracellular IP₃ receptor (IP₃R) (18–21). However, whether mTrp can serve as an appropriate model system for understanding I_CRAC activation is unresolved (18, 21, 22). In previous studies, we demonstrated that activity of I_min in i/o patches is potentiated by addition of IP₃R-enriched microsomes as predicted by an I_min-IP₃R conformational coupling model (16). More recently, we discovered that anti-PIP₂ antibody (PIP₂Ab) sensitizes I_min to IP₃ activation and proposed an I_min-IP₃R-PIP₂ functional coupling model based on these findings (17). In parallel with our results, a potential role of PIP₂ in trp-like (trpl) channel activation has been recently demonstrated in Sf9 cells (23). The I_min-IP₃R-PIP₂ coupling model can adequately explain activation of I_min channels by direct action of PLC but not the activation of I_CRAC channels resulting from Ca²⁺ store depletion (4–6).

A number of critical questions related to a depletion-activated Ca²⁺ influx pathway remain unanswered. Most importantly, do store-depletion and PLC-dependent pathways activate the same or a different channel type? To answer this question, we compare the effects of PLC-linked agonist UTP, Ca²⁺ pump inhibitor thapsigargin (Tg), and Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) on plasma membrane Ca²⁺ channel activity in patch–clamp experiments performed with human carcinoma A431 cells. We conclude that PLC activation and depletion of intracellular Ca²⁺ stores activate the same Ca²⁺ channel in A431 cells. We found that the conductance and selectivity properties of the store-operated channel in A431 cells are identical to the properties of I_min and somewhat different from the properties of I_CRAC channels described in Jurkat T cells (5–7). Thus, we will refer to store-operated channels in A431 cells as I_CRACL (“crac-like”). We also concluded that PIP₂ plays a role in modulation of I_CRACL activity.

Materials and Methods

Electrophysiological Recordings.

Human carcinoma A431 cells (Cell Culture Collection, Institute of Cytology, St. Petersburg, Russia) were kept in culture, as described elsewhere (12). For patch–clamp experiments, cells were seeded onto coverslips and maintained in culture for 1–3 days before use. Single-channel currents were recorded by using the cell-attached and i/o configuration of the patch–clamp technique (24). Currents filtered at 500 Hz were recorded with a PC-501A patch–clamp amplifier (Warner Instruments, Hamden, CT) with a conventional resistance feedback in the headstage (10 GΩ). The currents were digitized at 2.5 kHz. For data analysis and presentation, currents were additionally digitally filtered at 100 Hz.

NP_o was determined by using the following equation: NP_o = 〈I〉/i, where 〈I〉 and i are the mean channel current and unitary current amplitude, respectively. 〈I〉 was estimated from the time integral of the current above the baseline, and i was determined from current records and all-point amplitude histograms. Data were collected from current records after channel activity reached steady state. Because channel activity was transient and displayed significant fluctuations, we used NP_o collected during 30 s of maximal activity (NP_o^max₃₀) as a standard way to compare open channel probability among different experiments. Average NP_o^max₃₀ values of channel activity from several experiments are presented in the text and on the figures as mean ± SEM. The pipette solution contained (in mM): 105 BaCl₂, 105 CaCl₂, or 140 NaCl as indicated, and 10 Tris/HCl (pH 7.4). In cell-attached experiments, the bath solution contained 140 mM KCl and 2 mM CaCl₂ to nullify the cell's resting potential. For BAPTA-AM loading, 100 μM BAPTA-AM and 1 μM Tg were added to the bath solution containing (in mM): 140 KCl, 5 NaCl, 10 Hepes/KOH, and 2 EGTA (pH 7.4). For i/o experiments, patches were excised into the standard intracellular solution containing (in mM): 140 K glutamate, 5 NaCl, 1 MgCl₂, 10 Hepes/KOH, 1.13 CaCl₂, and 2 EGTA (pCa 7, pH 7.4), with or without IP₃ as indicated. The cell-attached and i/o recordings were performed at −70 mV holding potential. All experiments were carried out at room temperature (22–24°C).

Materials.

Monoclonal anti-PIP₂ antibody (PIP₂Ab) (25) was from PerSeptive Biosystems (Framingham, MA), and monoclonal anti-PIP antibody (PIPAb) was from Assay Designs (Ann Arbor, MI). PIP₂Ab and PIPAb were reconstituted in PBS (titer 1:1,500), diluted 1:100 by intracellular solution and used for chamber perfusion. Hepes, UTP, and Tg were from Sigma; EGTA was from Fluka Chemie AG (Buchs, Switzerland). IP₃ and BAPTA-AM were from Calbiochem.

Results

Exposure to Extracellular UTP Sensitizes I_min to IP₃ Activation.

When cell-attached (c/a) recordings of I_min in A431 cells were performed in control recording conditions, the channel activity was very low with NP_o^max₃₀ equal to 0.08 ± 0.06 (n = 12) (Fig. 1 a and c). After patch excision in bath solution containing 2.5 μM IP₃, moderate activity of I_min in i/o patches was observed with NP_o^max₃₀ equal to 0.86 ± 0.2 (n = 12) (Fig. 1 a and c). Similar behavior of I_min channels in c/a and i/o configurations has been described (10–12, 17). As we previously reported, addition of 100 μM UTP or 10 μM bradykinin to the solution bathing A431 cells leads to activation of PLC-coupled receptors and an increase in I_min activity in c/a patches to NP_o^max₃₀ of 0.7–1.0 (12). When 100 μM UTP was included in the pipette solution, significantly higher I_min channel activity was observed with NP_o^max₃₀ equal to 1.5 ± 0.17 (n = 33) (Fig. 1b). With either bath (12) or pipette (Fig. 1b) UTP application, activity of I_min was transient and resulted in channel inactivation within several minutes. After patch excision into intracellular solution containing 2.5 μM IP₃, very high levels of I_min channel activity were observed (Fig. 1b). On an average, I_min channel NP_o^max₃₀ increased from 1.31 + 0.17 (c/a) to 2.91 + 0.23 (i/o) in this series of experiments (n = 9) following patch excision (Fig. 1c).

Figure 1.

Sensitization of I_min to IP₃ by extracellular UTP. (a) Plot of I_min open channel probability (NP_o) in cell-attached (c/a) patch recorded in control conditions and in i/o patch from the same cell in the presence of 2.5 μM IP₃. The NP_o was averaged over 1-s intervals and plotted vs. time in the experiment. Mean NP_o ^max ₃₀ was 0.08 in c/a and 0.88 in i/o for the experiment shown. Data are representative of 12 experiments. (b) Same plot as in a for the experiment with 100 μM UTP in the pipette. Mean NP_o^max₃₀ was 1.5 in c/a and 3.41 in i/o for the experiment shown. Data are representative of nine experiments. (c) The summary plot of I_min open channel probability in c/a (open bars) and i/o (closed bars) recordings performed in control conditions (n = 12, left) or in the presence of 100 μM UTP in the pipette (n = 9, right). I_min activity is represented as NP_o^max₃₀ (mean ± SEM). (d) NP_o^max₃₀ of I_min channels in i/o experiments at IP₃ concentrations as indicated measured with 100 μM UTP in the pipette (▴), in the presence of PIP₂Ab (●) and in control conditions (○). Average data at each IP₃ concentration are shown as mean ± SEM (n ≥ 6). Smooth curve, best fit the data obtained with UTP in the pipette by using the equation NP_o^max₃₀ = (NP_o^max₃₀)^max [IP₃]^nH/([IP₃]^nH + K_app^nH), the values of parameters are in the text. Dashed lines, fit to the similar data obtained in control conditions (curve on the right) and in the presence of PIP₂Ab (curve on the left). The data for control conditions and in the presence of PIP₂Ab are taken from ref. 17.

To gain insight into the mechanism responsible for the unusually high activity of I_min channels in i/o recordings observed in the experiments with UTP in the pipette (Fig. 1 b and c), we determined the sensitivity of I_min activation by IP₃ when 100 μM of UTP was included in the pipette solution. In all experiments of this series, we waited until I_min activity in c/a patches subsided before the patch excision. A single IP₃ concentration in the 0.05–2.5 μM range was tested in each experiment to avoid IP₃-induced I_min desensitization (12). Fitting the Hill equation to the data (Fig. 1d, ▴) yielded an apparent affinity (K_app) of 0.15 μM IP₃, maximal NP_o^max₃₀ (NP_o^max) of 3.33, and a Hill coefficient (n_H) of 0.83 (Fig. 1d, curve). When similar experiments were performed in control recording conditions, sensitivity of I_min to IP₃ activation was much lower (K_app = 0.51 μM IP₃, NP_o^max = 0.87, n_H = of 1.05) (17) (Fig. 1d, ○, and dashed line on the right). The dramatic increase in I_min apparent affinity for IP₃ and in NP_o^max induced by exposure to UTP in the pipette quantitatively matches with the effects exerted by PIP₂Ab on I_min (K_app = 0.08 μM IP₃, NP_o^max = 3.21, n_H = of 0.8) (17) (Fig. 1d, ●, and dashed line on the left) and on the IP₃R (26). We reasoned that synergistic actions of extracellular UTP and intracellular IP₃ in our experiments (Fig. 1) result from UTP receptor stimulation of PLC which decreases PIP₂ levels in the patch. Reduction of PIP₂ levels leads to an increase in the apparent affinity of IP₃R for IP₃ (26) and in the potency of IP₃ to activate I_min.

I_min Is the I_CRACL Channel Activated by Depletion of Intracellular Ca²⁺ Stores.

I_CRAC currents can be activated in cells without PLC activation as a result of intracellular Ca²⁺ store depletion following exposure to Ca²⁺-ATPase inhibitor Tg or intracellular Ca²⁺ chelators BAPTA and EGTA (4, 5). The experiments described in the previous section support the I_min-IP₃R-PIP₂ coupling model (17). This model explains activation of I_min channels by direct action of PLC but not the activation of I_CRAC channels by Ca²⁺ store depletion. Does depletion of Ca²⁺ stores activate the same channel as activation of PLC? To answer this question, we evaluated effects of Tg on Ca²⁺ channel activity in patch–clamp experiments. As in our previous studies (12), addition of 1 μM Tg to the bath had only minimal effect on I_min activity when compared with control conditions, with NP_o^max₃₀ equal to 0.11 ± 0.03 (n = 9) (Fig. 2a; also see Fig. 4 a and h). In contrast to these results, if 1 μM Tg was included in the pipette, active I_min channels were observed following a short delay after patch formation, with NP_o^max₃₀ equal to 1.7 ± 0.24 (n = 18) (Figs. 2b and 4 b and h). We interpret this delay as the time needed for depletion of submembrane Ca²⁺ stores by Tg entering the cell from the pipette.

Figure 2.

Activation of I_min channels by Tg. (a) Ca²⁺ channel current traces in c/a patches recorded in the presence of 1 μM Tg in the bath solution. The fragments of current records are shown on the bottom on expanded time scale. The unitary current amplitude in used recording conditions (−70 mV membrane resting potential) is −0.18 pA. (b) Same as in a with 1 μM Tg in the pipette. (c) Same as in a with 100 μM BAPTA-AM and 1 μM Tg in the bath.

Figure 4.

Role of PIP₂ in I_CRACL modulation. (a) Plot of I_CRACL open channel probability (NP_o) in c/a patch recorded with 1 μM Tg in the bath and in i/o patch from the same cell in the presence of 2.5 μM IP₃. The NP_O was averaged over 1-s intervals and plotted vs. time in the experiment. Mean NP_o^max₃₀ was 0.07 in c/a and 0.66 in i/o for the experiment shown. Data are representative of nine experiments. (b) Same plot as in a for the experiment with 1 μM Tg in the pipette. Mean NP_o^max₃₀ was 1.62 in c/a and 0.09 in i/o for the experiment shown. Data are representative of 20 experiments. (c) Same plot as in a for the experiment with 100 μM BAPTA-AM and 1 μM Tg in the bath. Mean NP_o^max₃₀ was 0.48 in c/a and 0 in i/o for the experiment shown. Data are representative of nine experiments. (d) Same plot as in b, but with patch excision within 30 s after I_CRACL activation. Data are representative of four experiments. (e) I_CRACL channel current traces in c/a patches recorded in the presence of 1 μM Tg in the pipette solution followed by i/o current recordings in the presence of 2.5 μM IP₃ and PIP₂Ab as shown. Data are representative of seven experiments. (f) Same as in e with 100 μM BAPTA-AM and 1 μM Tg in the bath. Data are representative of four experiments. (g) Same as in e with the order of PIP₂Ab and IP₃ additions to i/o patch reversed. Data are representative of five experiments. (h) The summary plot of I_CRACL open channel probability in c/a (open bars) and i/o (closed bars) recordings performed in the presence of 1 μM Tg in the bath (n = 9, left) or in the presence of 1 μM Tg in the pipette (n = 20, right). I_CRACL activity is represented as NP_o^max₃₀ (mean ± SEM).

One potential explanation of different effects caused by bath and pipette applications of Tg is Ca²⁺-induced inactivation of I_min. From comparison of I_min rundown kinetic with Ca²⁺ or Ba²⁺ as a current carrier, we previously concluded that I_min is likely to undergo Ca²⁺-induced inactivation process (11). Massive Ca²⁺ release from the stores resulting from bath application of Tg may quickly inactivate I_min, but if Tg is included only in the pipette, Ca²⁺ leak is much slower, and I_min inactivation may be reduced or decelerated. To test this hypothesis, we clamped Ca²⁺ concentration in A431 cells by loading them with the membrane-permeable Ca²⁺ chelator BAPTA-AM. Bath application of 0.1 mM BAPTA-AM by itself resulted in I_min activity in 9 of 15 experiments. In six remaining experiments, application of Tg to BAPTA-loaded cells evoked I_min channel activity. To simplify experimental procedure, we combined application of Tg and BAPTA-AM to the bath, which resulted in I_min channel activity in 7 of 10 experiments (Figs. 2c and 4c). From these results, we concluded that the low potency of Tg in the bath to activate I_min in our previous experiments (Fig. 2a) (12) mostly likely results from Ca²⁺-dependent inactivation of I_min.

Activation of I_min by depletion of intracellular Ca²⁺ stores with Tg and BAPTA-AM (Fig. 2 b and c) reinforces the idea that I_min and I_CRAC may in fact be the same channels (17). To test this idea further and in the absence of molecular information and specific blockers, we resorted to comparison of I_min and I_CRAC single-channel properties. The divalent single-channel conductance of I_CRAC channels in Jurkat T cells has been estimated to be 24 fS from the noise analysis (6), and the monovalent single-channel conductance has been measured at 40 pS with Na⁺ as a current carrier (7). It has also been demonstrated that the permeability of I_CRAC to Ca²⁺ is higher than for Ba²⁺ (6, 27). With 105 mM divalent cations in the pipette, the store-operated channels in A431 cells were equally permeable to Ca²⁺ and Ba²⁺ (Fig. 3), displayed a single-channel current amplitude of −0.18 pA at −70 mV membrane potential (Figs. 2 and 3) and a single-channel conductance of about 1 pS (Fig. 3). Thus, conductance properties of these channels are identical to the properties of I_min channels activated by UTP (in c/a) or by IP₃ (in i/o) (12). We also demonstrated that the open probability of store-operated channels in A431 cells is strongly dependent on the membrane potential (Fig. 3), in line with the properties of I_min (12). Using 140 mM Na⁺ as a current carrier, we determined that store-depletion activated channels in A431 cells displayed the main conductance level of −0.56 pA at −70 mV membrane potential and the corresponding single channel conductance of 6 pS (Fig. 3), which is several-fold smaller than conductance of I_CRAC channels in Jurkat T cells in similar ionic conditions (7). From these results, we concluded that the store-depletion activated Ca²⁺ current in A431 is carried by I_CRAC-like (I_CRACL) channels, which are identical to the previously described I_min channels (12). In the remaining section of the paper, these channels will be referred to simply as I_CRACL.

Figure 3.

Conductance properties of store-operated channels in A431 cells. (a) Store-operated channels in A431 cells, activated by the mixture of 100 μM BAPTA-AM and 1 μM Tg in the bath solution, were recorded in c/a mode with 105 mM Ba²⁺ (Left), 105 mM Ca²⁺ (Center), and 140 mM Na⁺ (Right) in the pipette solution at membrane potential as indicated. (b) Fit to the unitary current-voltage relationship of store-operated channels with Ba²⁺ (▾, n = 4–6), Ca²⁺ (▵, n = 4), Na⁺ (●, n = 3) yielded slope single-channel conductance of 1 pS for Ca²⁺ and Ba²⁺ and 6 pS for Na⁺. (c) Open channel probability of store-operated channels (NP_o^max₃₀) expressed as a function of membrane potential. Data from six independent experiments in c/a mode with 105 mM Ba²⁺ as a current carrier were averaged at each membrane potential (▾). (b and c) The average values are shown as mean ± SEM, unless the size of the error bars is smaller than the size of the symbols.

PIP₂ Is a Modulator of I_CRACL.

When activated by UTP (Fig. 1b) or Tg (Fig. 4b), I_CRACL channel activity was transient, with channels typically lasting between 2 and 5 min. Loading of A431 cells with BAPTA-AM dramatically extended the period of Tg-induced I_CRACL activity, effectively preventing I_CRACL inactivation (Fig. 4c). Thus, we concluded that the Ca²⁺-dependent mechanism plays a major role in I_CRACL inactivation, similar to the previous studies of I_CRAC (27, 28). To get additional insight into the mechanisms of I_CRACL inactivation, we evaluated responses of I_CRACL channels to IP₃ in i/o patches. With 1 μM Tg in the bath, normal activation of I_CRACL channels by 2.5 μM IP₃ was observed in i/o patches (Fig. 4 a and h), similar to control experiments (Fig. 1a). However, exposure to 1 μM Tg in the pipette, which initially resulted in I_CRACL activation, eventually led to channel inactivation and greatly diminished activity of IP₃-gated I_CRACL channels in i/o mode (Fig. 4b). On average, I_CRACL channel activity in i/o patches with Tg in the pipette was reduced to NP_o^max₃₀ equal to 0.11 ± 0.03 (n = 20) (Fig. 4h). Tg-induced loss of I_CRACL channel sensitivity to activation by IP₃ developed in time. Indeed, when patches were excised within 30 s from the initial channel activation, substantial I_CRACL channel activity in i/o patches was initially observed in the presence of 2.5 μM IP₃ in 1 of 4 experiments (Fig. 4d). Although loading the cells with BAPTA-AM almost completely removed I_CRACL inactivation in c/a mode (Fig. 4c), the channels in these experiments were also unresponsive to IP₃ in i/o mode (Fig. 4c). Thus, following exposure to Tg, patch excision lead to a loss of I_CRACL responsiveness to IP₃, even in the absence of Ca²⁺-dependent inactivation.

Inclusion of UTP in the pipette resulted in sensitization of I_CRACL channels to IP₃ (Fig. 1 c and d), which we concluded was related to PLC-dependent reduction in PIP₂ levels in the patch (see above). What if depletion of Ca²⁺ stores, which leads to a loss of I_CRACL sensitivity to IP₃ in i/o patches (Fig. 4 b–d and h), increases the fraction of PIP₂-tethered IP₃R-I_CRACL complexes? To test this hypothesis, we analyzed the effect of PIP₂Ab on I_CRACL in i/o patches taken from cells exposed to Tg in the pipette or to the BAPTA-AM/Tg mixture in the bath. Although I_CRACL was rendered sensitive to IP₃ as a result of prolonged patch exposure to Tg, addition of PIP₂Ab restored I_CRACL channel activity (Fig. 4e), with NP_o^max₃₀ = 2.73 ± 0.3 (n = 7) (Fig. 4h). Similar results were obtained in the experiments (n = 4) where I_CRACL channels were initially activated by a BAPTA-AM/Tg mixture in the bath (Fig. 4f). The observed effect was specific for PIP₂Ab, as addition of PIPAb had no effect on I_CRACL channel activity in control experiments (n = 5). Similar to our previous results (17), PIP₂Ab alone did not induce channel activity in these conditions, but instead greatly potentiated the ability of IP₃ to activate the I_CRACL (Fig. 4g). The experiments with PIP₂Ab support the hypothesis that, following exposure to Tg and store-depletion, all I_CRACL-IP₃R complexes in the patch are shifted to the PIP₂-tethered state. In the absence of Ca²⁺-induced inactivation, I_CRACL channels in I_CRACL-IP₃R-PIP₂ complexes remain active as long as store is depleted but do not respond to IP₃.

Discussion

PLC-Dependent and Store-Operated Pathways of I_CRACL Activation.

Our results lead us to conclude that both PLC-linked and Ca²⁺ store-operated Ca²⁺ entry pathways in A431 cells are in fact supported by the same Ca²⁺ channel, with single-channel properties identical to the properties of the previously described I_min channel (12). Similar to I_min, the store-operated channels in A431 cells are equally permeable to Ca²⁺ and Ba²⁺ and display a divalent single channel conductance of 1 pS. Monovalent single-channel conductance of these channels is 5.5–6 pS with 140 mM Na⁺ as a current carrier, which is several-fold smaller than single-channel conductance of I_CRAC channels in Jurkat T cells measured in similar ionic conditions (40 pS) (7). To account for the observed differences in conductance and selectivity properties, we called the store-operated channel in A431 cells I_CRACL (I_CRAC-like). Ca²⁺ channels activated by depletion of intracellular stores in A431 cells were previously described (29). However, these channels are clearly distinct from I_CRACL as they display higher permeability to Ba²⁺ than to Ca²⁺ (16 pS at 160 mM Ba²⁺ and 2 pS at 200 mM Ca²⁺), not permeable to Na⁺, not voltage-dependent, and do not respond to IP₃ in i/o patches (29). Therefore, these channels constitute an alternative depletion-activated Ca²⁺ influx pathway in A431 cells. We have not observed channels described by Luckhoff and Clapham (29) in our experiments, most likely because of variability between different A431 clones or effects of culture conditions on channel expression. In some patches on A431 cells, we observed nonselective cation permeable channels with large conductance, which were clearly distinct from the I_CRACL. These channels did not respond to IP₃ or Tg, and the patches containing these channels were discarded.

What is a mechanism of I_CRACL activation? From the present results and from our previous work on I_min, we conclude that I_CRACL channels in A431 cells are conformationally coupled to intracellular IP₃R and can be activated: (i) by changes in the IP₃R receptor conformation on IP₃ binding (16); (ii) by direct cleavage of I_CRACL-IP₃R-tethered PIP₂ by PLC (17); and (iii) by the store-operated mechanism as in the conformational coupling mechanism originally proposed by Irvine (ref. 30) (present results). Gating of I_CRACL-IP₃R complexes by IP₃ probably accounts for the low background channel activity in resting cells (Fig. 1a) (endogenous IP₃ level is estimated at 40–100 nM in unstimulated cells; ref. 31), and for the substantial activity of I_CRACL channels in excised patches in the presence of 2.5 μM IP₃ (Fig. 1a). Cleavage of IP₃R-tethered PIP₂ by PLC is likely to be responsible for activation of I_CRACL channels by UTP in the pipette in our experiments (Fig. 1b). The activation of I_CRACL channels by Tg in the pipette (Fig. 2b) and by BAPTA-AM/Tg in the bath (Fig. 2c) results from the IP₃R conformational changes on intracellular Ca²⁺ store depletion. In physiological conditions, stimulation of cells by agonist leads to PLC activation, increase in IP₃ levels, and depletion of Ca²⁺ stores. Therefore, an additive or even synergistic action of three different pathways of I_CRACL activation in cells is expected in response to application of agonist in situ. Similar to I_CRAC (27, 28), I_CRACL channels are under strong negative inhibitory control by cytosolic Ca²⁺, which normally leads to a transient nature of I_CRACL activity (Figs. 1b and 4b). Loading A431 cells with BAPTA removes Ca²⁺-dependent inactivation and dramatically increases the duration of I_CRACL activity (Fig. 4c).

Role of PIP₂ as a Modulator of I_CRACL Channels.

Our data also suggest that PIP₂ may play a role of I_CRACL modulator by regulating a dynamic equilibrium between I_CRACL-IP₃R and I_CRACL-IP₃R-PIP₂ complexes (Fig. 5 Left). Following exposure to UTP, activation of PLC and cleavage of PIP₂ in the patch, the majority of I_CRACL channels are shifted to PIP₂-free I_CRACL-IP₃R state (Fig. 5 Top), as manifested by NP_o^max₃₀ = 3 in i/o patches with 2.5 μM IP₃ in these experiments (Fig. 5 Top Right) compared with NP_o^max₃₀ = 0.86 in control patches (Fig. 5 Right). Depletion of the stores with Tg or BAPTA appears to shift the equilibrium in the opposite direction, with all of I_CRACL channels driven to I_CRACL-IP₃R-PIP₂ complexes (Fig. 5 Bottom). I_CRACL channels in these experiments were unresponsive to IP₃ in i/o patches with NP_o^max₃₀ = 0.1 (Fig. 5 Bottom Right) but responded essentially at the maximal level (NP_o^max₃₀ = 2.7) to a combination of 2.5 μM IP₃ and PIP₂Ab (Fig. 4d). Despite loss of sensitivity to activation by IP₃, I_CRACL channels in I_CRACL-IP₃R-PIP₂ complexes remain active in c/a mode (but not in i/o mode, for reasons that need to be further investigated) as long as stores are depleted and I_CRACL inactivation is prevented by chelating Ca²⁺ (Fig. 4c). Possible mechanisms responsible for the store-dependent shift toward a PIP₂-occupied state of the IP₃R may include physical rearrangement of mobile Ca²⁺ stores (32), changes in local PIP₂ levels in the patch (33), or an increase in IP₃R affinity for PIP₂ following Ca²⁺ stores depletion. Additional experiments will be needed to discriminate between these possibilities.

Figure 5.

Model of I_CRACL conformational coupling to IP₃R and modulation by PIP₂. I_CRACL-IP₃R and I_CRACL-IP₃R-PIP₂ complexes exist in equilibrium in resting cells (Left). Background I_CRACL channel activity in cell-attached patches in resting cells results from endogenous IP₃ (40–100 nM) (31) activating I_CRACL-IP₃R complexes. Exposure of patches excised from the resting cells to 2.5 μM IP₃ leads to elevated I_CRACL channel activity in i/o configuration (Right). Exposure to UTP in the pipette triggers cleavage of IP₃R-tethered PIP₂ and direct activation of I_CRACL-IP₃R-PIP₂ complexes in cell-attached patches as previously proposed (17). The shift from I_CRACL-IP₃R-PIP₂ to I_CRACL-IP₃R complexes (Top) explains high activity of I_CRACL channels in i/o patches in the presence of 2.5 μM IP₃ (Top Right). Exposure to Tg in the pipette or to BAPTA-AM/Tg in the bath causes depletion of local Ca²⁺ stores and activation of I_CRACL channels by means of conformational coupling mechanism (30). Depletion of Ca²⁺ stores by some unknown mechanism promotes formation of I_CRACL-IP₃R-PIP₂ complexes (Bottom), which leads to the loss of I_CRACL sensitivity to activation by 2.5 μM IP₃ in excised patches (Bottom Right). Despite loss of sensitivity to activation by IP₃, I_CRACL channels in I_CRACL-IP₃R-PIP₂ complexes remain active as long as stores are depleted and I_CRACL inactivation is prevented by chelating Ca²⁺ with BAPTA. The model drawing is adapted from ref. 47.

Conformational Coupling Model of I_CRACL Activation.

I_CRACL-IP₃R association is likely to involve direct binding of the IP₃R amino-terminal region to the I_CRACL protein, similar to mTrp-IP₃R association (21). Interestingly, the same amino-terminal region of IP₃R also includes specific IP₃ (34, 35) and PIP₂ (36) binding sites. Thus, ligand-induced conformational changes of the IP₃R amino-terminal region can be transmitted directly to the I_CRACL channel. The store-operated I_CRACL activation is likely to involve an IP₃R-associatied endoplasmic reticulum resident Ca²⁺-binding protein, such as calreticulin (37–39), which serves as a sensor of intraluminal Ca²⁺. Additional signaling components are likely to be recruited to the I_CRACL-IP₃R complex via actions of a modular adaptor protein, such as mGluR1/IP₃R-binding protein Homer in neuronal cells (40), the Syk/Btk/Grb2/PLCγ-binding protein BLNK in B lymphocytes (41), or the trp/PKC/PLC-binding protein inaD in Drosophila photoreceptors (42). The actin cytoskeleton may also play an important role in correct spatial arrangement of required signaling components (43–45). In chicken B lymphocytes, removal of all three IP₃R isoforms by genetic means had no effect on Tg-induced Ca²⁺ influx (46), in apparent conflict with the conformational coupling model of I_CRACL activation in A431 cells (Fig. 5). From these results, we conclude that the B lymphocytes must have an additional or alternative Ca²⁺ influx pathway, coupled to Ca²⁺ store depletion by means of IP₃R-independent mechanism that may involve a global “diffusible messenger.” Additional functional studies with B lymphocytes will be required for its detailed characterization.

Abbreviations

PLC

phospholipase C

I_CRAC

Ca²⁺ release activated channel

I_CRACL

I_CRAC-like

IP₃

inositol 1,4,5-triphosphate

IP₃R

IP₃ receptor

PIP₂

phosphatidylinositol (1,4,5)-bisphosphate

c/a

cell-activated

i/o

inside-out

Tg

thapsigargin

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate