An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes
2002; Springer Nature; Volume: 21; Issue: 17 Linguagem: Inglês
10.1093/emboj/cdf467
ISSN1460-2075
AutoresGuillermo Vázquez, Barbara J. Wedel, Gary S. Bird, Suresh K. Joseph, James W. Putney,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle2 September 2002free access An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes Guillermo Vazquez Guillermo Vazquez Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Barbara J. Wedel Barbara J. Wedel Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Gary St J. Bird Gary St J. Bird Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Suresh K. Joseph Suresh K. Joseph Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA, 19103 USA Search for more papers by this author James W. Putney Jr Corresponding Author James W. Putney Jr Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Guillermo Vazquez Guillermo Vazquez Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Barbara J. Wedel Barbara J. Wedel Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Gary St J. Bird Gary St J. Bird Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Suresh K. Joseph Suresh K. Joseph Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA, 19103 USA Search for more papers by this author James W. Putney Jr Corresponding Author James W. Putney Jr Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA Search for more papers by this author Author Information Guillermo Vazquez1, Barbara J. Wedel1, Gary St J. Bird1, Suresh K. Joseph2 and James W. Putney 1 1Calcium Regulation Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences—NIH, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC, 27709 USA 2Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA, 19103 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4531-4538https://doi.org/10.1093/emboj/cdf467 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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). Figure 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. Download figure Download PowerPoint 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 addition 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. Figure 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. Download figure Download PowerPoint 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. Figure 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. Download figure Download PowerPoint 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. Figure 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. Download figure Download PowerPoint 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. Figure 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. Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint 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. Figure 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. Download figure Download PowerPoint 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, Icrac (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 predomin
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