An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

Abstract

We examined the roles of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP₃R isoforms (IP₃R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba²⁺ than does capacitative calcium entry. This cation entry is absent in IP₃R-KO cells. Expression of the type-3 IP₃R (IP₃R-3) in the IP₃R-KO cells rescued not only agonist-dependent release of intracellular Ca²⁺, but also Ba²⁺ influx following receptor stimulation. Similar results were obtained with an IP₃R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP₃R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca²⁺ release or receptor-dependent Ba²⁺ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP₃R.

Introduction

Activation of the phospholipase C pathway in non-excitable cells results in the generation of inositol 1,4,5-trisphosphate (IP₃), which stimulates the release of Ca²⁺ store in the endoplasmic reticulum (Berridge, 1993). This release of Ca²⁺ is generally associated with an increase in Ca²⁺ entry across the plasma membrane that can serve to replenish stores or to contribute to Ca²⁺-dependent signaling. In the majority of instances, this entry of Ca²⁺ appears to be signaled through a poorly understood mechanism that is initiated by depletion of the Ca²⁺ stores (Putney, 1986), a process known as capacitative calcium entry (CCE) or store-operated calcium entry (Putney, 1997a).

Non-CCE mechanisms have been described in a variety of cell types (Fasolato et al., 1993; Byron and Taylor, 1995; Shuttleworth and Thompson, 1996; Carroll and Peralta, 1998; Broad et al., 1999). The mechanisms controlling Ca²⁺ channel activation in such instances are not known, but may involve other second messengers, for example arachidonic acid. One second messenger that has been considered as a signal for activating plasma membrane Ca²⁺ entry is IP₃ acting on plasma membrane IP₃ receptors (IP₃R). In fact, a number of studies have provided biochemical evidence for the presence of IP₃R in the plasma membrane of cells (Guillemette et al., 1988; Fujimoto et al., 1992; Khan et al., 1992a,b, 1996; Feng and Kraus-Friedmann, 1993; Bush et al., 1994; Mayrleitner et al., 1995; Quinton and Dean, 1996; El-Daher et al., 2000; Tanimura et al., 2000). However, there is little physiological evidence that these receptors play a significant role in Ca²⁺ signaling at the plasma membrane. The one notable exception is vertebrate and invertebrate olfactory neurons, for which there is considerable functional evidence for the presence of plasma membrane IP₃-gated channels (Cunningham et al., 1993; Fadool and Ache, 1994; Okada et al., 1994; Kashiwayanagi, 1996; Lischka et al., 1999; Munger et al., 2000). A role for the IP₃R type-3 (IP₃R-3) has been proposed as responsible for capacitative Ca²⁺ entry in some cell types (Putney, 1997b), but it is clear this cannot be the case in all cell types (Wang et al., 2001).

Both T and B lymphocytes have been shown, by immunostaining, to possess plasma membrane IP₃R, and these receptors have been suggested to play a role in apoptosis (Khan et al., 1992a,b, 1996). We have investigated the role of IP₃R in plasma membrane calcium fluxes, utilizing the DT40 B lymphocyte cell line. This is an excellent model for investigating the function of IP₃R in B cell signaling because of the availability of a mutant DT40 line lacking all three IP₃R isoforms (Sugawara et al., 1997). DT40 is a chicken B cell line expressing an IgM isotype B cell receptor (BCR) in the plasma membrane (Baba et al., 1985) that is linked to intracellular Ca²⁺ ([Ca²⁺]_i) signaling through phospholipase Cγ (PLCγ) activation (Buerstedde and Takeda, 1991; Sugawara et al., 1997). PLCγ activation results in conversion of phosphatidylinositol 4,5-bisphosphate into IP₃, which then releases Ca²⁺ from thapsigargin-sensitive intracellular stores by interacting with specific IP₃R in the endoplasmic reticulum (Berridge, 1993). This release results in transient elevation of cytosolic calcium concentration and is followed by a more sustained Ca²⁺ entry from the outside, which is mediated, at least in part, by the store-operated, CCE pathway (Putney, 1986; Berridge, 1995; Parekh and Penner, 1997; Barritt, 1999; Sugawara et al., 1997). In the present work we demonstrate that there is also a non-capacitative pathway for calcium entry in DT40 B lymphocytes, activated through the BCR. This entry pathway, while not mediated by store depletion, requires a functional IP₃R. In light of previous work providing biochemical evidence for IP₃R in the plasma membrane of B cells and other cell types, we suggest that this entry of divalent cations may result from direct gating of plasma membrane IP₃R by IP₃.

Results

As shown in Figure 1, activation of the BCR in DT40 B lymphocytes, in the absence of extracellular Ca²⁺, results in an oscillatory [Ca²⁺]_i signal, presumably due to cyclical release of Ca²⁺ from intracellular stores. In the current study, Ca²⁺ responses to BCR activation were observed in 60–70% of cells tested (n = 195). Restoration of extracellular Ca²⁺ to the responsive cells revealed a stimulated entry of Ca²⁺ in 80% of the BCR-responsive cells. Both the release and entry of Ca²⁺ appear to depend on IP₃R in some manner, because both phases of the response are lost in cells lacking IP₃R (Sugawara et al., 1997), and both phases are completely blocked by the potent membrane permeant IP₃R inhibitor, xestospongin C (Gafni et al., 1997; Figure 1, top). Release of Ca²⁺ by the SERCA inhibitor, thapsigargin, and the resulting CCE, were not inhibited by xestospongin C (Figure 1, bottom).

Fig. 1. The IP₃R inhibitor xestospongin C effectively suppresses both BCR-induced Ca²⁺ release and calcium entry in DT40 cells B lymphocytes, but does not affect thapsigargin-induced Ca²⁺ release or entry. DT40 cells were incubated for 45 min in the presence of 25 µM xestospongin C. Cells were then bathed in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody (top) or 2 µM thapsigargin (bottom), as indicated. Ca²⁺ (1.5 mM) was added where indicated. In this experiment, relatively higher ratio values were obtained as compared with other experiments due to the use of a different Ca²⁺ measuring system; in this series (and for experiments in Figure 3), a photon counting system was used, while in the other series, an imaging system was used. Black trace: control (no xestospongin C); gray trace, 25 µM xestospongin C. Shown are representative traces from three independent experiments.

The function of IP₃R in the Ca²⁺ entry phase could be due to their role in the release of store Ca²⁺ and activation of store-operated channels, or alternatively there may be some other, distinct mechanism for regulation of Ca²⁺ by IP₃R. If the latter were the case, then one prediction is that BCR stimulation should activate an entry in addi tion to that, dependent solely on Ca²⁺ store depletion. Furthermore, this response should be absolutely dependent on the presence of IP₃R. This appears to be the case, as shown by results in Figure 2. In these experiments, we took advantage of the fact that under our experimental conditions, the store-operated Ca²⁺ entry pathway involves either no detectable Ba²⁺ entry or, in some experiments, a small Ba²⁺ entry (Vazquez et al., 2001; data not shown; Figure 2A). However, unlike thapsigargin-activated CCE, BCR stimulation results in a substantial entry of Ba²⁺ entry (Figure 2B). This clearly indicates that BCR-dependent cation entry involves not only store-operated channels, but also a population of somewhat less selective cation channels requiring signals other than, or in addition to, store depletion.

Fig. 2. BCR activation, but not store depletion, induces Ba²⁺ entry in DT40 B lymphocytes. (A) Fura-2-loaded WTDT40 cells were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin in order to deplete intracellular Ca²⁺ stores. After cytosolic Ca²⁺ returned to basal levels, Ba²⁺ (10 mM) was added to the medium. A representative trace from at least five independent experiments is shown. (B) Wild-type (WTDT40) or IP₃R-KO (broken line) DT40 cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from at least five independent experiments are shown.

In experiments utilizing Ba²⁺ to assess entry, 102 of 127 anti-IgM responsive cells showed Ba²⁺ entry. Note that in a previous publication, Venkatachalam et al. (2001) failed to observe agonist-activated Ba²⁺ entry. This difference is most likely due to the fact that Venkatachalam and colleagues used 1 mM Ba²⁺, while in our experiments the concentration of Ba²⁺ was 10 mM.

The failure to observe cation entry in all anti-IgM-responsive cells could result from rapid desensitization of the receptor–phospholipase C pathway. In fact, desensitization of the B cell antigen receptor may occur with as few as 5% of the receptors occupied, resulting in blockade of the signaling pathways derived from receptor activation by a mechanism apparently involving a short-term action of PKC (Vilen et al., 1997). There was no detectable Ba²⁺ entry in those cells unable to respond to BCR crosslinking (68 of 68 cells). Importantly, the receptor-dependent Ba²⁺ entry was never observed in IP₃R knockout cells (IP₃R-KO, a variant lacking the three IP₃R isoforms, IP₃R-1, -2 and -3) (Figure 2B), indicating that the receptor-activated cation influx is dependent on the presence of IP₃R rather than depletion of Ca²⁺ stores.

In the remaining experiments, we took advantage of the apparent higher Ca²⁺ selectivity of the endogenous store-operated channels in order to assess BCR-dependent cation (Ba²⁺) entry into the cells without contribution from receptor activated endogenous CCE channels (see for example Vazquez et al., 2001). In addition, as Ba²⁺ is a poor substrate for endoplasmic reticulum or plasma membrane calcium pumps (Vanderkooi and Martonosi, 1971; Yamaguchi et al., 1989) and thus is not readily cleared from the cytoplasm, it provides a reliable way to monitor unidirectional cation entry that avoids potential complications derived from changes in cellular Ca²⁺ metabolism or Ca²⁺-dependent regulation of the cation channels (Byron and Taylor, 1995).

The failure of BCR stimulation to activate entry in IP₃R-KO cells indicates the importance of the IP₃R in this response, but does not rule out contributions of other elements of the BCR signaling pathway. Thus, we next transiently transfected DT40 cells with the human M5 muscarinic receptor, which would activate a different PLC, PLCβ, through a G-protein rather than tyrosine kinase-dependent mechanism. As shown in Figure 3, carbachol stimulation of the M5-expressing cells resulted in a rapid and transient cytosolic calcium rise (12 of 17 EYFP⁺ cells), which rapidly declined to basal levels in ∼2 min; addition of Ba²⁺ to the external medium resulted in significant cation entry, which was not observed in those cells unable to respond to muscarinic stimulation. Again, this receptor-dependent Ba²⁺ entry was never observed in IP₃R-KO cells transfected with the M5 receptor. These results indicate that the cation entry route following receptor activation was related to IP₃ production and IP₃R expression, but not linked to the type of receptor eliciting the signal.

Fig. 3. Activation of the G-protein-coupled M5 muscarinic receptor results in stimulation of Ba²⁺ entry in wild-type but not in IP₃R-KO DT40 cells. Ba²⁺ influx was measured in single Fura-2-loaded wild-type (WTDT40) or IP₃R-KO (dotted line) DT40 cells transfected with the human M5 muscarinic receptor. The cells were maintained in nominally Ca²⁺-free medium, exposed to 100 µM carbachol, and then Ba²⁺ (10 mM) was added where indicated. The relatively higher ratio values in this experiment are due to the use of a different Ca²⁺ measuring system; in this series, a photon counting system was used, while in the other series, an imaging system was used. Representative traces from three independent experiments are shown.

We next considered the possibility that activation of Ba²⁺ entry following BCR crosslinking could be the consequence of activation of Ca²⁺-activated non-selective cation channels due to the transient cytosolic Ca²⁺ rise that follows IP₃-mediated Ca²⁺ mobilization from inner stores. In order to demonstrate clearly the dependence of the Ba²⁺ entry on BCR stimulation, we utilized the following protocol. Both wild-type (WTDT40) and IP₃R-KO cells were treated with thapsigargin to discharge intracellular stores completely. Ba²⁺ was then added and, as shown previously, no detectable entry of Ba²⁺ occurred. Subsequently, anti-IgM was added, and in the WTDT40, but not in the IP₃R-KO cells, Ba²⁺ entry occurred almost immediately (Figure 4). Interestingly, with this protocol a greater proportion of the cells (67 of 71) exhibited significant cation entry upon BCR stimulation, probably due to the fact that Ba²⁺ entry is triggered almost as soon as the receptor pathway becomes activated, thus minimizing any possible desensitization of the receptor signaling route.

Fig. 4. BCR-dependent activation of Ba²⁺ entry in wild-type but not in IP₃R-KO DT40 cells. Fura-2-loaded wild-type (WTDT40) and IP₃R-KO DT40 cells were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin. After complete store depletion, Ba²⁺ (10 mM) was added to the medium where indicated, and 2 min later anti-IgM (5 µg/ml) was added. Representative traces from three independent experiments are shown.

One interesting point that can be taken from the protocol shown in Figure 4 is that the delay in Ba²⁺ entry is noticeably briefer than that for release following BCR activation. The average delay for the increase in Ba²⁺ entry after anti-IgM addition from the experiments in Figure 4 was 18 ± 10 s, while the delay for release from various protocols varied from 40 to 60 s. This is perhaps not surprising, if the entry is due to IP₃ formed and acting at the plasma membrane, while release results from an action of IP₃ at the endoplasmic reticulum.

To confirm that it is the IP₃R that is necessary for the BCR-dependent Ba²⁺ entry in DT40 cells, we attempted to restore this entry mechanism in IP₃R-KO cells by transient expression of the rat IP₃R-3. As predicted, stimulation of BCR signaling in IP₃R-3-expressing IP₃R-KO cells resulted not only in reappearance of the BCR-dependent, IP₃-mediated release of stored Ca²⁺, but also in a significant Ba²⁺ entry following receptor activation (21 of 26 IgM-responsive EYFP⁺ cells; Figure 5). Similarly, carbachol stimulation of IP₃R-KO cells transiently co-transfected with the M5 muscarinic receptor and the rat IP₃R-3 resulted in rapid release of calcium from stores followed by activation of the Ba²⁺ entry route in response to carbachol (data not shown). Neither the transient Ca²⁺ release nor the Ba²⁺ entry was detected in mock-transfected IP₃R-KO cells. These results indicate that in DT40 B lymphocytes, activation of the BCR signaling pathway results in generation of IP₃, which in turn activates not only the endoplasmic reticulum-located IP₃R that cause Ca²⁺ release from endogenous stores, but also a cation entry pathway in the plasma membrane strongly linked to IP₃R expression. Unlike the highly selective CRAC channels, the IP₃R is a relatively less selective cation channel more permeable to Ba²⁺ than to Ca²⁺ (Bezprozvanny and Ehrlich, 1994, 1995). Thus, the simplest explanation for this IP₃R-dependent entry is that it represents cation flux through plasma membrane IP₃R. Although this would appear to be the most straightforward explanation for the current observations, we cannot rule out an indirect regulation of a plasma membrane channel by IP₃R, as in the conformational coupling mechanism for TRP or store-operated channels (Irvine, 1990; Berridge, 1995; Kiselyov et al., 1998). In an attempt to resolve this issue we transiently expressed two different pore mutants of the IP₃R-3 into IP₃R-KO cells and tested their abilities to restore BCR stimulation of the Ba²⁺ entry pathway. It has recently been shown that the aspartic acid residue at position 2550 of the rat IP₃R-1 plays a critical role in cation permeation through the channel (Boehning and Joseph, 2000). This amino acid is located within a highly conserved sequence in the IP₃R (GVGD) that is homologous to the selectivity filter in potassium channels (Doyle et al., 1998; see diagram in Figure 6). Replacing this aspartate by alanine (D2550A) results in an IP₃R that is still able to bind IP₃ but devoid of Ca²⁺ release activity, i.e. rendering an impermeable channel. A conservative mutation in which the aspartate is replaced by a glutamic acid (D2550E) fully preserves the IP₃-induced Ca²⁺ release activity of the channel. These mutations do not alter the ability of the channels to express at high levels or to form oligomers (Boehning and Joseph, 2000). Unfortunately, we have not as yet successfully expressed the rat IP₃R-1 in DT40 IP₃R-KO cells. Thus, we engineered these same two point mutations in the equivalent aspartate residue at position 2477 in the rat IP₃R-3 (mutants D2477A and D2477E, respectively). As shown in Figure 6, the D2477A mutant, when expressed in IP₃R-KO cells, failed to restore either BCR-induced Ca²⁺ release or receptor-dependent Ba²⁺ entry. Heterologous expression of the D2477A IP₃R-3 mutant was confirmed by immunocytochemistry (Figure 6), and the extent of expression and localization of the mutant appeared to be identical to that of the wild-type IP₃R-3. Unfortunately, because of the small size of DT40 and the expression of IP₃R-3 on endoplasmic reticulum through the non-nuclear regions, we were not able to definitively localize the receptor to the plasma membrane by immunocytochemistry.

Fig. 5. Transient expression of an IP₃R restores agonist-induced Ba²⁺ entry in IP₃R-KO DT40 cells. Ba²⁺ influx was measured in Fura-2-loaded IP₃R-KO DT40 cells transfected with either the rat IP₃R-3 or its vector (Mock). The cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from three independent experiments are shown.

Fig. 6. Effect of single point mutations within the putative pore forming region of IP₃R-3 on restoration of agonist-induced Ba²⁺ entry in IP₃R-KO DT40 cells. The top of the figure shows an alignment of a region including the predicted pore of rat IP₃R-1–3 (the putative selectivity filter sequence is underlined). D2477 of rat IP₃R-3, which was mutated into glutamate (D2477E) or alanine (D2477A), corresponds to D2550 of rat IP₃R-1. In the experiment shown below, Ba²⁺ influx was measured in Fura-2-loaded IP₃R-KO DT40 cells transfected with either the D2477E (solid line) or the D2477A (broken line) mutants of the rat IP₃R-3 (see Materials and methods for details on the mutations). The cells were maintained in nominally Ca²⁺-free medium, exposed to 5 µg/ml anti-IgM antibody, and then Ba²⁺ (10 mM) was added where indicated. Representative traces from three independent experiments are shown. At the bottom, immunolocalization of transiently expressed rat IP₃R-3 (pCB6⁺ vector) in DT40 B cells is shown. IP₃R-KO cells untransfected, or transfected with the wild-type rat IP₃R-3 (WT-IP₃R), or the rat IP₃R-3 mutant D2477A (D2477A-IP₃R; see text for details) were incubated with a mouse anti-IP₃R-3 monoclonal antibody and then with the secondary Alexa Fluor 488-labeled anti-mouse IgG antibody. The fluorescence images were acquired with a Zeiss 410 confocal microscope.

Because we cannot be certain that depletion of stores is not required for activation of this pathway, we also tested the D2477A mutant in a protocol in which Ca²⁺ stores were first depleted by thapsigargin; again, no BCR stimulation of Ba²⁺ entry was observed (Figure 7). The inability of this mutant to restore receptor-stimulated, IP₃-mediated release of Ca²⁺ from intracellular stores is consistent with the expectation that replacement of the D2477 within the predicted selectivity region of the channel by a non-negatively charged amino acid would abrogate cation permeation through the channel pore. However, the D2477E mutant, in which the negative charge is preserved by replacing aspartate with glutamate, fully retained the phenotype exhibited by IP₃R-KO cells transiently expressing the wild-type IP₃R-3. Mutant or truncated IP₃R lacking functional pores are known to be fully capable of activating plasma membrane channels through the conformational coupling mechanism (Kiselyov et al., 1998, 1999). Thus, if the BCR-activated Ba²⁺-permeable pathway were mediated by plasma membrane cation channels conformationally coupled to IP₃R, then the D2477A IP₃R-3 mutant would be expected to gate the coupled Ba²⁺-permeable receptor-activated channel in the plasma membrane despite its inability to release calcium from the stores. However, only the D2477E mutant, with intact gating and permeation properties, was able to restore both the release and the cation entry pathways driven by BCR stimulation.

Fig. 7. The D2477A mutant of the rat IP₃R-3 does not restore BCR-dependent activation of Ba²⁺ entry in Ca²⁺-depleted, IP₃R-KO DT40 cells. Fura-2-loaded IP₃R-KO DT40 cells transfected with either the wild type or the D2477A mutant of the rat IP₃R-3 were incubated in nominally Ca²⁺-free medium and then exposed to 2 µM thapsigargin. After complete store depletion, Ba²⁺ (10 mM) was added to the medium where indicated, and 2 min later anti-IgM (5 µg/ml) was added. Solid line, wild-type IP₃R; dashed line, D2477A IP₃R. Representative traces from three independent experiments are shown.

Discussion

WTDT40 B lymphocytes co-express three subtypes of IP₃R: IP₃R-1, -2 and -3 (Sugawara et al., 1997), which functionally interact to produce the oscillatory Ca²⁺ pattern observed with BCR activation by antigen- or antibody-dependent crosslinking (Miyakawa et al., 1999). A variant of these cells has been generated in which the genes coding for the three IP₃R isoforms have been disrupted by homologous recombination (Sugawara et al., 1997). Such IP₃R-KO cells respond to PLC-coupled receptors with generation of IP₃, but they do not generate PLC-linked cytosolic Ca²⁺ signals. It has been shown previously that store-operated Ca²⁺ entry occurs normally in both wild-type and IP₃R-KO DT40 cells following passive depletion of endogenous stores with thapsigargin (Sugawara et al., 1997; Broad et al., 2001; Ma et al., 2001). This endogenous store-operated pathway is highly selective for Ca²⁺ and exhibits minimal permeability to Ba²⁺ or Sr²⁺ (Figure 2A; see also Vazquez et al., 2001; Venkatachalam et al., 2001). Consistent with this conclusion, which is based on fluorescence measurements, Prakriya and Lewis have recently demonstrated that the store-operated entry in DT40 involves the highly selective calcium current, I_crac (Prakriya and Lewis, 2001).

The results of the current study demonstrate the presence of a novel mechanism for BCR-activation of divalent cation entry across the plasma membrane of B cells that requires fully functional IP₃R, and apparently does not involve a conformational coupling mechanism. The most likely scenario is that in DT40 B lymphocytes, stimulation of the BCR-associated signaling machinery results in generation of IP₃, which, in addition to inducing calcium release from stores through activation of endoplasmic reticulum-located IP₃R, also activates plasma membrane located IP₃R. Ca²⁺ entry through this pathway may contribute to BCR-mediated Ca²⁺ signaling, or, possibly, cation fluxes may modify store-operated Ca²⁺ entry in other ways. For example, if substantial cation fluxes were to result in membrane depolarization, then store-operated entry might actually be inhibited. This would be consistent with the observation of Hashimoto et al. (2001), who, using a membrane potential-sensitive dye, observed membrane depolarization and inhibition of CCE by BCR activation in DT40 cells. Thus, it would be useful to demonstrate the presence of an agonist-activated membrane current with properties expected of an IP₃R. However, probably due to the small size of this current, we have not as yet succeeded in identifying a non-capacitative whole-cell current activated by agonist in either WTDT40 or IP₃R-3 transfected IP₃R-KO cells.

Although not the major focus of this study, the experiments involving transfection of IP₃R-KO cells with IP₃R-3 revealed interesting patterns of Ca²⁺ signaling in the release phase as well. Miyakawa et al. (1999) generated different combinations of single IP₃R-KO DT40 cells and evaluated the contribution of each of the IP₃R subtypes normally expressed in these cells (IP₃R-1, -2 and -3) to the oscillatory Ca²⁺ pattern observed upon BCR activation. Interestingly, those studies suggested that only mutants expressing the IP₃R-2 exhibited BCR-dependent Ca²⁺ oscillations comparable to the wild-type cells, whereas mutants expressing either IP₃R-1 or IP₃R-3 responded with a monophasic Ca²⁺ transient. In the present studies, we observed that transient expression of the rat IP₃R-3 in IP₃R-KO DT40 cells is sufficient to restore an oscillatory pattern for the Ca²⁺ response to BCR stimulation (26 of 42 EYFP⁺ cells). As differences in the expression level of the IP₃R isoforms may affect the Ca²⁺ signaling pattern, it is possible that transient overexpression of the IP₃R-3 could account for such a discrepancy. Our observations are also inconsistent with the suggestion from Hagar et al. (1998) that cells expressing predominantly type 3 IP₃R would not support Ca²⁺ oscillations due to a lack of Ca²⁺-dependent inhibition of the channel. In this context, the ability of the IP₃R-KO cells transiently and solely expressing the rat IP₃R-3 to respond to BCR stimulation with clearly defined cytosolic Ca²⁺ oscillations could be taken as indirect evidence for biphasic regulation of the IP₃R-3 upon cytoplasmic Ca²⁺ concentration; however, it is also possible that cyclical modulation of PLC activity could underlie these oscillations.

As discussed above, a number of publications have raised the issue of plasma membrane IP₃R, but, with the exception of invertebrate olfactory neurons, the significance of these observations is unclear (Fasolato et al., 1994). Confusion undoubtedly arises because in some instances, IP₃ appears to be capable of regulating store-operated channels (Vaca and Kunze, 1995; Kiselyov et al., 1998; Zubov et al., 1999); this probably represents a conformational coupling mechanism, rather than the presence of plasma membrane IP₃R. In the current study, however, the use of Ba²⁺ as a surrogate for Ca²⁺, as well as findings presented in Figures 3–5, clearly demonstrate that DT40 B cells express a novel entry pathway that is distinct from the store-operated pathway, does not appear to involve conformational coupling and is dependent on the presence of a cation-permeable IP₃R. Taken together with previous immunological evidence for the presence of IP₃R in the B cell plasma membrane (Khan et al., 1996), it seems likely that this IP₃R-dependent pathway reflects Ba²⁺ entry through plasma membrane IP₃R cation channels.

Non-CCE mechanisms have been described in a variety of cell types (Fasolato et al., 1993; Byron and Taylor, 1995; Shuttleworth and Thompson, 1996; Carroll and Peralta, 1998; Broad et al., 1999). One of the hallmarks of non-capacitative pathways is their greater permeability of Ca²⁺ surrogates, as compared with Ca²⁺ (Byron and Taylor, 1995). In at least two different cell types, the receptor-activated, non-capacitative Ca²⁺ entry can be activated by arachidonic acid (Shuttleworth, 1997; Broad et al., 1999), leading to speculation that a receptor-regulated phospholipase A₂ may be involved. However, in HEK293 cells, we previously found (Luo et al., 2001) that the non-capacitative Ca²⁺ entry activated by low concentrations of muscarinic agonists was inhibited by 2-aminoethoxydiphenyl borane, a compound that inhibits both store-operated channels, as well as IP₃R (Maruyama et al., 1997; Braun et al., 2001; Broad et al., 2001; Dobrydneva and Blackmore, 2001; Gregory et al., 2001; Iwasaki et al., 2001; Ma et al., 2001; Prakriya and Lewis, 2001). It is thus tempting to speculate that plasma membrane IP₃R may underlie, or at least contribute to, non-capacitative Ca²⁺ entry in cell types other than the B lymphocyte. This issue should be the focus of investigation in future studies in a variety of Ca²⁺-regulated systems.

Materials and methods

Cell culture, transfection and measurement of intracellular calcium

The immortalized chicken B lymphocyte cell line, DT40 (RIKEN Cell Bank No. RCB1464), and its mutant variant lacking all three IP₃R types (RIKEN Cell Bank No. RCB1467) were kindly provided by Dr Tomohiro Kurosaki (Department of Molecular Genetics, Kansai Medical University). Cell culture and handling for intracellular calcium measurements using the calcium-sensitive dye Fura-2 were as described previously (Vazquez et al., 2001).

DT40 cells were transiently transfected by electroporation (300 V, 500 µF) with either the rat wild-type IP₃R-3 in pCB6⁺ (provided by Dr Graeme Bell, University of Chicago, Chicago, IL), its mutants (see below) or vector alone (mock-transfected cells), along with pEYFP-C1 (Clontech, Palo Alto, CA) as a transfection marker. In some experiments, cells were transfected with the human M5 muscarinic receptor (kindly provided by Dr Lutz Birnbaumer, National Institutes of Environmental and Health Sciences, Research Triangle Park, NC); the corresponding cDNA was subcloned into an expression vector (lcf201, provided by Drs Jean-Marie Buerstedde/Hiroshi Arakawa, Heinrich-Pette-Institute, University of Hamburg, Germany) under the control of the chicken β-actin promoter for expression in DT40 cells. Cells were assayed 18–30 h post-transfection. Fluorescence measurements were performed on EYFP⁺ cells selected by their green fluorescence when excited at 485 nm. The fluorescence intensity of multiple Fura-2-loaded DT40 cells was monitored with a CCD camera-based imaging system (Universal Imaging) mounted on a Zeiss Axiovert 35 inverted microscope equipped with a Zeiss 40× (1.3 NA) fluor objective. A Sutter Instruments filter changer enabled alternative excitation at 340 and 380 nm, while the emission fluorescence was monitored at 510 nm with a Paultek Imaging camera (model PC-20) equipped with a GenIISys intensifier (Dage-MTI, Inc.). The images of multiple cells collected at each excitation wavelength were subsequently processed using the MetaFluor software (Universal Imaging Corp., West Chester, PA) to provide ratio images. In some experiments, responses of single cells were analyzed by utilizing a photomultiplier-based system, as described previously (Vazquez et al., 2001) (for example, Figures 1 and 3). Cells transfected with vector alone (mock-transfected cells) were used in parallel as controls. All experiments were performed at room temperature. The data are expressed as a ratio of Fura-2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm (F340/F380).

Mutagenesis

The cDNA of rat IP₃R-3 (Blondel et al., 1993) in pCB6⁺ (kindly provided by Dr Graeme Bell) was used as a template for mutagenesis. D2477A and D2477E point mutations were generated using the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. Mutations were verified by DNA sequencing.

Immunocytochemistry for IP₃R-3

DT40 cells attached to coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% NP-40 in phosphate-buffered saline. Non-specific sites were blocked with 3% bovine serum albumin in TBST (Tris–HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20) at room temperature for 30 min, and the cells were then incubated with anti-IP₃R-3 monoclonal antibody (Transduction Laboratories, Lexington, KY) at 1:500 dilution. Secondary antibody was the Alexa fluor 488-labeled goat anti-mouse IgG antisera. The fluorescence images were acquired with a Zeiss LSM410 confocal laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) using an argon–krypton laser and excitation at 488 nm through a 40× (1.2 NA, water immersion) objective lens (optical slice thickness 1.5 µm).