Agonist-induced Ca²⁺ entry determined by inositol 1,4,5-trisphosphate recognition

Abstract

It has been considered that Ca²⁺ release is the causal trigger for Ca²⁺ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP₃R), the lack of Ca²⁺ entry in response to receptor activation is attributed to the absence of Ca²⁺ release. We reveal in this article that IP₃R recognition of IP₃ determines agonist-induced Ca²⁺ entry (ACE), independent of its Ca²⁺ release activity. In DT40 IP₃R^–/– cells, endogenous ACE can be rescued with type 1 IP₃R mutants (both a ΔC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca²⁺ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP₃R-dependent manner without Ca²⁺ store release. Conversely, ACE cannot be rescued with mutant IP₃Rs lacking IP₃ binding (both the Δ90–110 and R265Q IP₃-binding site mutants). We conclude that an IP₃-dependent conformational change in the IP₃R, not endoplasmic reticulum Ca²⁺ pool release, triggers ACE.

Ca²⁺ transients elicited in response to cell surface receptor activation by neurotransmitters, hormones, and other molecular messengers are major messengers of intracellular communication (1). Stimulation of G protein-coupled receptors, tyrosine kinase receptors, and nonreceptor tyrosine kinases activate phospholipase C (PLC), catalyzing the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP₂) into the second-messenger molecules: inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ mediates rapid Ca²⁺ store release by activating IP₃ receptors (IP₃Rs) in the endoplasmic reticulum (ER), whereas DAG activates protein kinase C (PKC) (2). After this initial Ca²⁺ release phase, external Ca²⁺ enters through plasma membrane channels, providing a secondary and more prolonged Ca²⁺ signal (1), a phenomenon designated here as agonist-induced Ca²⁺ entry (ACE) (3).

To date, the molecular identity of these Ca²⁺ entry channels as well as their coupling mechanism remain unknown, although several mechanisms have been proposed. Intracellular Ca²⁺ release through the IP₃R could trigger ACE by means of capacitative Ca²⁺ entry (CCE) (4), which can be activated in a PLC-independent manner (3) by the ER Ca²⁺ pump blocker thapsigargin or the Ca²⁺ ionophore ionomycin (4, 5). In this scheme, the luminal drop in ER Ca²⁺ activates “store-operated” Ca²⁺ channels in the plasma membrane, although the basis for this coupling mechanism is entirely unknown. Although the release activity of IP₃Rs may mediate Ca²⁺ entry, others have suggested that IP₃Rs play a conformational role in the coupling process (6, 7). Also, DAG may directly initiate ACE, because DAG can activate overexpressed “canonical” transient receptor potential Ca²⁺ entry channels (TRPC) (8).

We recently demonstrated a functional distinction between ACE and CCE based on the requirement of the former for PLC-γ, in a lipase-independent manner (3). However, it is unclear whether endogenous ACE is an integrated process accounting for both receptor and store-operated channel activation. At the molecular level, it is unclear whether IP₃R-mediated Ca²⁺ release triggers ACE and/or whether a conformational alteration of the IP₃R is sufficient for ACE. Evidence for the activation by Ca²⁺ pool emptying alone (and the dispensability of the IP₃R for Ca²⁺ entry) includes the ability of thapsigargin or ionomycin, but not PLC activation, to stimulate Ca²⁺ entry in the DT40 triple IP₃R knockout cell line, a form of B lymphocytes devoid of any IP₃R (9). These interpretations assume that ACE and CCE are functionally overlapping mechanisms. In the present study, we demonstrate that IP₃ recognition by the IP₃R but not the receptor's Ca²⁺ channel activity is required for activation of endogenous ACE.

Materials and Methods

Culture of Cells. Rat PC12 cells (passage numbers 6–15), human embryonic kidney (HEK)293 cells, rat aortic smooth muscle A7r5 cells (passage numbers 10–25), and DT40 chicken B lymphocyte IP₃R^–/– cells were cultured as described (3, 9, 10).

Expression Protocols. DT40, HEK293, PC12, and A7r5 were transfected as described (3). For experiments shown in Figs. 3 and 4, 5 μg of yellow fluorescent protein (YFP) cDNA ± 20 μg of IP₃R cDNA was used for transfection.

Fig. 3.

Differential rescue by type 1 WT IP₃R of Ca²⁺ release and entry in DT40 IP₃R^–/– B cells. Free Ca²⁺ measurements were made in DT40 IP₃R^–/– knockout cells transfected with either YFP plus WT IP₃R (WT; A–C) or YFP alone (D). Ca²⁺ pools were released in cells by 3 μg/ml anti-IgM (arrow) in nominally Ca²⁺-free medium followed by replacement with anti-IgM and normal 1 mM Ca²⁺ medium (bar). Representative traces are individual single-cell responses.

Fig. 4.

Channel-deficient type 1 IP₃Rs rescue Ca²⁺ entry in DT40 IP₃R^–/– B cells, whereas IP₃-binding-deficient type 1 IP₃Rs do not. Free Ca²⁺ measurements were made in DT40 IP₃R^–/– knockout cells transfected with either YFP plus WT IP₃R (WT; A), YFP plus ΔC-terminal IP₃R mutant (ΔC; B), YFP plus Δ90–110 IP₃R mutant (Δ90–110; C), YFP plus D2550A IP₃R mutant (D2550A; D), YFP plus R265Q IP₃R mutant (R265Q; E), or YFP alone (F). Ca²⁺ pools were released in cells by 3 μg/ml anti-IgM (arrow) in nominally Ca²⁺-free medium followed by replacement with anti-IgM and normal 1 mM Ca²⁺ medium (bar). DT40 IP₃R^–/– knockout cells, transfected as above, were prepared for immunocytochemistry and visualized by confocal microscopy, and they appear directly below their respective Ca²⁺ trace. (A–F) The Left images corresponds to YFP expression, and the Right images corresponds to IP₃R staining. All traces are averages of ≈15 cells because the mutants Δ90–110 or R265Q IP₃Rs do not respond and ΔC or D2550A only displays the NRE response.

Ca²⁺ Imaging. Ca²⁺ measurements were as described (11). Fura-2/acetoxymethyl ester loading was for 25 min at 20°C for DT40 cells, 1 h at 20°C for PC12 cells, 25 min at 20°C for HEK293 cells, and 30 min at 20°C for A7r5 cells. Transfected enhanced YFP served as the transfection marker and was detected at excitation wavelength 485 nm. Resting Ca²⁺ levels in cell lines were similar, 100–200 nM, and cells with higher basal levels were excluded from data collection because these cells tend to have constitutive Ca²⁺ entry. All measurements shown are representative of a minimum of three and, in most cases, a larger number of independent experiments. For the population studies in Fig. 1, YFP-transfected cells were used to control for experiments in HEK293 cells transfected with YFP plus the sarcoplasmic ER Ca²⁺ ATPase (SERCA)-2b. Functional rescue of Ca²⁺ responses in the DT40 IP₃R^–/– cells was totaled from five separate experiments with six conditions each: (i) YFP alone, 0 out of 241 cells (0% rescue, 0.0 SEM); (ii) WT IP₃R, 49 out of 261 cells (18.7% rescue, 0.36 SEM); (iii) ΔCIP₃R, 58 out of 230 cells (25% rescue, 1.62 SEM); (iv) Δ90–110 IP₃R, 1 out of 239 cells (0.4% rescue, 0.19 SEM); (v) D2550A IP₃R, 67 out of 258 cells (25.9% rescue, 0.64 SEM); and (vi) R265Q IP₃R, 0 out of 255 cells (0% rescue, 0.0 SEM).

Fig. 1.

Ca²⁺ entry in the absence of IP₃R-mediated Ca²⁺ release. Free Ca²⁺ measurements were made in YFP-transfected PC12, HEK293, and A7r5 cells. (A) Ca²⁺ pools were released by bradykinin (BK, 1 μM; arrow) in nominally Ca²⁺-free medium followed by replacement with bradykinin and normal 1 mM Ca²⁺ medium (bar). (B) Ca²⁺ pools were released by CCH (100 μM; arrow) in nominally Ca²⁺-free medium followed by replacement with CCH and normal 1 mM Ca²⁺ medium (bar). (C) Ca²⁺ pools were released by vasopressin (Vaso, 1 nM; arrow) in nominally Ca²⁺-free medium followed by replacement with vasopressin and normal 1 mM Ca²⁺ medium (bar). Average traces are representative of 30–50 cells, and the subpopulation traces are representative single cells extracted from that average.

Immunohistochemistry. Immunohistochemistry was as described in ref. 3.

IP₃R Mutagenesis. Δ90–110 IP₃R was constructed with two rounds of PCR by using overlapping 40-mers in which the bases 270–330 were omitted.

Antibodies and Reagents. Plasmids were obtained from the following sources: enhanced YFP vector cDNA was from Clontech; IP₃R WT type 1, ΔC terminus, D2550A, and R265Q vectors were from Suresh Joseph (Thomas Jefferson University, Philadelphia, PA); carbachol (CCH), bradykinin, and vasopressin were from Sigma; Fura-2/acetoxymethyl ester and goat-anti rabbit Alexa-568 were from Molecular Probes; anti-chicken IgM (supernatant, M-4 clone) was from Southern Biotechnology Associates; and polyclonal anti-IP₃R was from Affinity BioReagents (Golden, CO).

Results

Many studies, including our own, have shown that Ca²⁺ entry is coincident with IP₃R-mediated Ca²⁺ release (3, 10, 12, 13). Such studies are almost exclusively based on average responses within large numbers of cells, despite the risks of interpreting Ca²⁺ signals from populations (14). In the present study, we have compared Ca²⁺ release and Ca²⁺ entry in individual cells. We measured single-cell receptor-mediated Ca²⁺ responses to bradykinin receptors in PC12 cells (neuronal origin), muscarinic receptors in HEK293 cells (kidney origin), and vasopressin receptors in A7r5 cells (vascular smooth muscle-derived). Ca²⁺ release was induced by the addition of agonist in nominally Ca²⁺ free media (Fig. 1, arrows). Subsequent addition of agonist with 1 mM extracellular Ca²⁺ induces an increase in cytosolic Ca²⁺, resulting from ACE (Fig. 1, bars). In averaged responses from multiple (30–50) cells, Ca²⁺ release coincides with ACE in each of the three cell types examined (Fig. 1, leftmost traces). However, analysis of the single-cell responses comprising these averages reveals three subpopulations of Ca²⁺ signals. Overall, among the three cell types, 62–72% of individual cells display both Ca²⁺ release and Ca²⁺ entry (RE), 17–20% of cells exhibit Ca²⁺ release with little to no Ca²⁺ entry (RNE), and 10–17% of cells manifest little to no Ca²⁺ release yet substantial Ca²⁺ entry (NRE) (Fig. 1 and Table 1). In all three cell types, constitutive Ca²⁺ entry is a rare event (<1% of cells), and a similarly small population of cells do not show any Ca²⁺ response (these cells were excluded from the population analyzed).

Table 1. Frequency of various Ca²⁺ entry responses to agonist stimulation.

Cell/agonist	n	RE, %	RNE, %	NRE, %
PC12/1 μM bradykinin	427	72.60	17.56	9.84
A7r5/10 nM vasopressin	208	62.98	20.19	16.83
HEK293/100 μM CCH	411	72.70	17.03	10.21

Ca²⁺ transients were collected for YFP-transfected PC12, A7r5, and HEK293 cells over five standardized experiments. These responses were categorized into their respective groups by a semiquantitative approach in which Ca²⁺ - release events <20 nM were considered little to no release and Ca²⁺ entry events <20 nM were considered little to no entry.

We wondered whether these frequencies could be manipulated by overexpressing SERCA-2b, which is known to increase IP₃-releasable Ca²⁺ stores in transfected cells (15, 16). Compared with control-transfected HEK293 cells (Table 1), the proportion of cells manifesting RE is reduced in SERCA-2b-transfected HEK293 cells (control, 72.7% vs. 59.82%; n = 131/219 cells) (data not shown). By contrast, the proportion of cells with RNE is markedly augmented (control, 17.03% vs. 39.73%; n = 87/219), and virtually no cells are detected in the NRE phenotype (control, 10.21% vs. 0.457%; n = 1/219). This result is inconsistent with the Ca²⁺ entry process being triggered by Ca²⁺ release, because SERCA-2b expression would have augmented ACE.

We characterized the relationship between Ca²⁺ release and entry in HEK293 cells by performing scatter analysis of the two parameters in response to the muscarinic receptor agonist CCH (100 μM) (Fig. 2). There is no correlation between extent of peak Ca²⁺ pool release and peak Ca²⁺ entry (n = 40; R = –0.05258). Thus, ACE in cells is not graded with respect to the magnitude of IP₃R-mediated Ca²⁺ release. This result agrees with the nonlinear activation of the store-operated Ca²⁺ current (I_crac) by IP₃ (14).

Fig. 2.

Agonist-induced Ca²⁺ release and entry are uncorrelated. Scatter plot of peak release size vs. peak entry magnitude from YFP-transfected HEK293 cells assayed by Ca²⁺ imaging. These single-cell traces were randomly chosen from experiments on 4 different days (10 per experiment), and absolute numbers reflect subtraction from baseline (n = 40, R = –0.05258).

Because our imaging assay reflects only whole-cell Ca²⁺ transients, we cannot exclude the possibility of subthreshold/local IP₃R-mediated Ca²⁺ release within the subpopulations of cells exhibiting NRE. Undetectable release from possibly specific “coupling pools” could trigger ACE and subsequent Ca²⁺-induced Ca²⁺ release through Ca²⁺ release channels. Under this paradigm, NRE (Fig. 1, rightmost traces) might be a mixture of Ca²⁺ release and Ca²⁺ entry. To overcome the difficulty in analyzing microdomains of local IP₃R-mediated Ca²⁺ release, we used the mutant DT40 chicken B lymphocyte cell line, which is deficient in all three genes for the IP₃R (IP₃R^–/–) (17). Stimulation of DT40 cells with the B cell receptor agonist anti-IgM (IgM) leads to a nonreceptor tyrosine kinase-linked activation of PLC-γ2 and subsequent production of IP₃ and DAG (3, 18). Introduction of Ca²⁺ reveals corresponding ACE as described (3, 9). Importantly, neither IP₃/DAG production nor thapsigargin- or ionomycin-induced activation of CCE differs between the WT DT40 and mutant IP₃R^–/– cells (9, 12). In rescue experiments, transfection of DT40 IP₃R^–/– cells with YFP and WT type 1 IP₃R (WT) (Fig. 3 A–C) restores PLC-dependent activation of ACE. Similar subpopulations of Ca²⁺ responses are seen in these rescued cells as in WT DT40 cells (data not shown). Moreover, even though mediated by PLC-γ, these subpopulations occur with comparable frequency as the PLC-β-stimulated responses in PC12, HEK293, and A7r5 cells (Fig. 1). Because YFP control DT40 IP₃R^–/– cells lack any Ca²⁺ release or ACE (Fig. 3D), we conclude that each of the three Ca²⁺ response subtypes requires the IP₃R. Thus, even the NRE response (Fig. 3C) is restored by IP₃R expression.

The DT40 IP₃R^–/– cells allowed us to assess directly the role of functionally modified IP₃Rs on ACE. We transfected the cells with two different IP₃R mutants: (i) C-terminal truncation mutant (ΔC), which binds IP₃ but cannot release Ca²⁺ because of defects in Ca²⁺ channel gating; or (ii) a mutant IP₃R lacking N-terminal amino acids 90–110 (Δ90–110), producing a channel with a ≈100-fold decrease in IP₃ binding (19). Compared with WT IP₃R rescue (Fig. 4A), expression of ΔC restores ACE without any Ca²⁺ release (Fig. 4B) (see Materials and Methods for the percentage of cells rescued), because the only response seen is the NRE response. In contrast, neither agonist-induced Ca²⁺ release nor Ca²⁺ entry is evident in cells transfected with Δ90–110 IP₃R, which cannot bind IP₃ (Fig. 4C). Because neither mutant channel can elicit intracellular Ca²⁺ release, the functional requirement for coupling to ACE appears to be IP₃ recognition by the IP₃R.

As the ΔC deletion involves removal of ≈300 amino acids, other receptor functions may have been altered. Additionally, the Δ90–110 IP₃R mutant still can bind IP₃, albeit with ≈100-fold lower affinity than WT. We addressed these issues in rescue experiments by using single point-mutant IP₃R constructs: (i) an IP₃R pore mutant (D2550A) lacking a critical aspartate-2550, mutation of which to alanine abolishes Ca²⁺ channel activity (20), or (ii) an IP₃R ligand-binding mutant (R265Q) lacking a critical arginine-265, mutation of which to glutamine abolishes IP₃ binding (21). The pore mutant (D2550A), like ΔC inactive channel preparation, restores Ca²⁺ entry but not release (NRE) (Fig. 4D), establishing that intracellular Ca²⁺ release is not required for entry. The deficient IP₃ binding mutant (R265Q), like the Δ90–110 mutant that cannot bind IP₃, displays neither Ca²⁺ release nor ACE (Fig. 4E).

D2550A rescue fails to support agonist-induced Ba²⁺ (1 mM) entry, fitting with the ion selectivity of IgM-activated WT DT40 cells (ref. 9 and data not shown). Vazquez et al. (12) reported that a pore-deficient mutant of type 3 IP₃R did not rescue agonist-induced Ba²⁺ (10 mM) entry in the DT40 IP₃R^–/– cells. We used type 1 IP₃R constructs and measured ACE. If IP₃R specificity (type 1 vs. type 3) does not underlie this difference, then we would conclude that the channel activity rescued by D2550A expression is Ca²⁺-specific.

In control experiments, we assessed the expression of DT40 IP₃R^–/– cells transfected with YFP alone, YFP plus WT IP₃R, or YFP plus mutant IP₃Rs by using a rabbit polyclonal antibody against an N-terminal IP₃R epitope and YFP as a marker for transfected cells. Confocal immunocytochemistry with goat anti-rabbit Alexa-568 secondary antibodies confirms the expression of the IP₃R constructs in transfected DT40 IP₃R^–/– cells [Fig. 4 A–E, YFP (Left) and anti-IP₃R (Right)]. In summary, our IP₃R mutational analysis establishes that ACE requires functionally active IP₃Rs and depends on IP₃ recognition but is independent of Ca²⁺ release activity.

Discussion

The present study shows that, unlike CCE, ACE activation does not require ER Ca²⁺ release. Rather, ACE requires recognition of IP₃ by the IP₃R, irrespective of any Ca²⁺ release activity. These conclusions are supported by several key findings. First, single-cell Ca²⁺ measurements reveal subpopulations of Ca²⁺ responses (RE, RNE, and NRE), and the magnitude of Ca²⁺ release does not correlate with the magnitude of ACE. Second, in DT40 IP₃R^–/– cells devoid of ACE, restoration of WT IP₃R expression rescues ACE in the three modes described, illustrating the requirement of the IP₃R for each response type. Third, ACE is rescued in the DT40 IP₃R^–/– cells by two distinct Ca²⁺ release-deficient IP₃R mutants but not by two different ligand-binding mutants. Thus, the IP₃R is coupled to ACE independently of its Ca²⁺ release activity, supporting the notion that conformational changes in the IP₃R, subsequent to IP₃ binding, gate endogenous Ca²⁺ entry channels.

Our findings on the requirement of IP₃ for ACE are consistent with studies showing that the IP₃R binds TRPC3 in HEK293 cells (22, 23) and that an N-terminal IP₃R fragment bound to IP₃ is sufficient to gate TRPC3 in reconstituted vesicles (22, 24). Moreover, the IP₃-dependence on ACE accords with experiments in the DT40 PLC-γ^–/– cells, in which a lipase-inactive PLC-γ mutant rescues ACE only in the presence of receptor-generated IP₃ (3). Our findings also complement observations of Penner and colleagues (25) that repetitive subthreshold CCH application stimulates ACE independently of Ca²⁺ release in RBL-2H3-M1 cells.

DAG, The other product of phosphatidylinositol 4,5-bisphosphate (PIP₂) degradation, also has been proposed as a physiological activator of ACE, given that it can stimulate overexpressed TRPC3, -6, and -7 channels, even in the absence of IP₃Rs (8, 9). Conversely, recent work reveals DAG-induced PKC activation as an inhibitor of endogenous ACE as well as overexpressed TRPC channels (26). In our experiments, endogenous ACE is not demonstrable in DT40 IP₃R^–/– cells, even though these cells contain active PLC and can produce DAG (9, 26). Thus, if DAG is an activator of Ca²⁺ entry, it is unlikely to function alone. Alternatively, IP₃ and DAG may function coordinately in an activation–deactivation loop. Recently, Delmas et al. (27) demonstrated that G protein-coupled receptors signal differently to specific microdomains of IP₃Rs in rat sympathetic neurons. By using overexpressed TRPC1 and TRPC6, these authors correlated Ca²⁺ release with TRPC1 activation and lack of Ca²⁺ release with TRPC6 (i.e., DAG activation). Our findings suggest that, although both TRPC1 and TRPC6 respond differentially to DAG, both still may be regulated by the IP₃R, with the activation of TRPC1 merely being coincident with Ca²⁺ release.

Our findings support a model whereby Ca²⁺ entry is elicited by an IP₃R pool in which no release occurs but not by an IP₃R pool in which only release occurs. This interpretation is supported by overexpression experiments of SERCA-2b in HEK293 cells that decrease RE and NRE but augment the RNE phenotype. Accordingly, the preferred coupling mode is presumably a functional IP₃-bound IP₃R in a nonfunctional ER Ca²⁺ pool. Alternatively, but less likely, overactive expressed SERCA-2b might scavenge Ca²⁺ as it enters the cell, thereby obscuring our measurements. Compatible with the former hypothesis, expression of ΔC or D2550A IP₃R, that can bind IP₃ but not release, augments ACE. Our model fits with suggestions of Parekh et al. (14), that distinct IP₃-sensitive ER stores function in release and entry modes, and Delmas et al. (27), who demonstrated IP₃R-signaling microdomains in neurons. As discrete processes, Ca²⁺ release and Ca²⁺ entry may be dynamically regulated through changes in the movement of IP₃Rs into or out of functional ER pools, generating microdomains that could respond differentially to various receptor stimuli and Ca²⁺-signaling functions.

Expression of WT IP₃Rs in the DT40 IP₃R^–/– rescues the NRE response, as does the pore mutant. If coupling occurs within a nonfunctional ER pool, then Ca²⁺ release by WT IP₃Rs would need to be rendered nonfunctional, at least in zones of Ca²⁺ entry. Modification of ER Ca²⁺ store content could be accomplished by proteins such as SERCA or by ER Ca²⁺ buffers (e.g., calsequestrin). Alternatively, but not mutually exclusive, a soluble truncated form of the IP₃R, containing the N-terminal IP₃ binding domain alone could couple IP₃ recognition to Ca²⁺ entry. Cells contain transcripts for IP₃Rs lacking Ca²⁺ channels (28), although it is not known whether these transcripts are translated into functional proteins. We suggest that such a protein may provide physiological signals for ACE, binding IP₃ and TRPCs but unable to release Ca²⁺. Overall, this mechanism resembles proposals of Irvine (6) and Berridge (7) over a decade ago for IP₃R-mediated conformational coupling.