Assessment of the Role of the Inositol 1,4,5-Trisphosphate Receptor in the Activation of Transient Receptor Potential Channels and Store-operated Ca2+ Entry Channels*

Hong-Tao MaDagger, Kartik VenkatachalamDagger, Hong-Sheng Li§, Craig Montell§, Tomohiro Kurosaki, Randen L. Patterson, and Donald L. Gill||

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, § Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and  Department of Molecular Genetics, Kansai Medical University, Moriguchi 570-8506, Japan

Received for publication, January 31, 2001, and in revised form, March 15, 2001


    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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The mechanism for coupling between Ca2+ stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP3R) was examined using the permeant InsP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP3R-mediated store release and carbachol-induced Sr2+ entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP3Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP3R-independent since InsP3Rs are dispensable for the light response. We used triple InsP3R knockout DT40 chicken B-cells to further assess the role of InsP3Rs in SOC activation. 45Ca2+ flux analysis revealed that although DT40 wild-type cells retained normal InsP3Rs mediating 2-APB-sensitive Ca2+ release, the DT40InsP3R-k/o cells were devoid of functional InsP3Rs. Using intact cells, all parameters of Ca2+ store function and SOC activation were identical in DT40wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC50) were identical. The results indicate that (a) the action of 2-APB on Ca2+ entry is not mediated by the InsP3R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Intracellular Ca2+ signals control diverse cellular functions ranging from short-term responses such as contraction and secretion to longer-term regulation of cell growth and proliferation (1, 2). The cytosolic Ca2+ signals generated in response to receptors are complex, involving two closely coupled components: rapid, transient release of Ca2+ stored in the endoplasmic reticulum (ER)1 followed by slowly developing extracellular Ca2+ entry (1, 3-7). G protein-coupled receptors and tyrosine kinase receptors, through activation of phospholipase C, generate the second messenger, inositol 1,4,5-trisphosphate (InsP3), that diffuses rapidly within the cytosol to interact with InsP3 receptors on the ER, which serve as Ca2+ channels to release luminal-stored Ca2+ and generate the initial Ca2+ signal phase (1, 4). The resulting depletion of Ca2+ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane, resulting in the slow activation of store-operated channels (SOCs), which mediate the process known as capacitative Ca2+ entry (3, 5-8). This second Ca2+ entry phase of Ca2+ signals serves to mediate longer term cytosolic Ca2+ elevations and provides a means to replenish intracellular stores (3, 5). Whereas receptor-induced generation of InsP3 and the function of Ca2+ release channels to mediate the initial Ca2+ signaling phase is well understood, the mechanism for coupling ER Ca2+ store depletion with Ca2+ entry remains a crucial but unresolved question (5-8).

Coupling to activate SOCs has been hypothesized to involve direct coupling between the ER and plasma membrane (9, 10), and evidence indicates that physical docking of ER with the plasma membrane may be involved in SOC activation (11-14). The mammalian TRP family of receptor-operated ion channels appears to share some operational parameters with SOCs (15, 16). Experiments reveal that the activation of TRPC3 channels like SOCs may require close coupling between the ER and plasma membrane (11, 17). Kiselyov et al. (18, 19) provide evidence that activation of human TRPC3 channels stably expressed in HEK293 cells occurs through a process involving endogenous InsP3 receptors. This activation appears to reflect a specific molecular interaction between the two channel proteins (20) analogous in some ways to the coupling between ryanodine receptors and Ca2+ entry channels in fast muscle triad junctions (1). Experiments reveal that the activation of both TRP channels and SOCs is prevented by the action of two different membrane-permeant InsP3 receptor antagonists, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB) (17, 21), providing further evidence for the involvement of the InsP3 receptor in coupling to activate both entry channel types. The analogy between the operation of SOCs and TRP channels is further strengthened by reports indicating that specific members of the TRP family of channels can operate in a store-dependent manner (18, 19, 22-27). However, there are also reports indicating that the ionic selectivity of TRP channels does not correspond with known SOC activities (28-31) and that TRP channels can operate independently of store depletion (31-37).

Given the uncertainty with respect to the function and coupling of store-operated Ca2+ entry, the current report focuses on two central questions: first, whether InsP3 receptors are involved in the operation of SOCs and TRP channels; and second, whether members of the TRP family of channels are operationally related to SOCs. The studies provide further assessment of the involvement of the InsP3 receptor in coupling to entry channels by comparing vertebrate and invertebrate TRP channel function with store-operated channels functioning in mammalian cells in which all three of the InsP3 receptor subtypes have been genetically deleted. The results indicate that although InsP3Rs do not mediate the action of 2-APB on Ca2+ entry, the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

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Culture of Cells-- Cells of both the wild-type DT40 chicken B cell line (DT40w/t) and triple InsP3 receptor knock-out cell line (DT40InsP3R-k/o) were cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine, as described previously (38). Cells of the T3-65 clone of HEK293 cells stably expressing the human TRPC3 channel were cultured as described previously (17, 21).

Measurement of Intracellular Calcium-- Cells grown on coverslips for 1 day were transferred to Hepes-buffered Krebs medium (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 1.2 mM KH2PO4, 11.5 mM glucose, 0.1% bovine serum albumin, 20 mM Hepes-KOH, pH 7.4) and loaded with fura-2/AM (2 µM) for 25 min at 20 °C. Cells were washed, and dye was allowed to de-esterify for a minimum of 15 min at 20 °C. Approximately 95% of the dye was confined to the cytoplasm as determined by the signal remaining after saponin permeabilization (39, 40). Fluorescence emission at 505 nm was monitored with excitation at 340 and 380 nm; Ca2+ measurements are shown as 340/380-nm ratios obtained from groups of 10-12 cells. Details of these Ca2+ measurements were described previously for the T3-65 clone of HEK293 cells (17, 21). Resting Ca2+ levels in the two DT40 cell lines were similar, ~100-130 nM, and in T3-65 HEK293 cells, resting Ca2+ was 50-100 nM. All measurements shown are representative of a minimum of three and, in most cases, a larger number of independent experiments.

Cell Permeabilization-- The procedures for cell permeabilization were as described earlier (41, 42). Briefly, suspensions of DT40 cells (1 × 106 cells/ml) were gently stirred and incubated with 0.0025% saponin in an intracellular-like medium (140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, and 10 mM Hepes-KOH, pH 7.0) at 37 °C until 95% permeabilization occurred (normally after 6-7 min). After permeabilization, cells were washed twice in saponin-free intracellular-like medium at 4 °C and kept cold before use in experiments. To avoid problems of lipid dilution of added hydrophobic compounds, the final cell concentration in all experiments was kept at exactly 5 × 105 cells/ml.

Calcium Flux Experiments-- Ca2+ flux measurements were conducted as previously described (42-44). The accumulation of 45Ca2+ into intracellular organelles was measured using permeabilized DT40 cells (5 × 105 cells/ml) maintained with gentle stirring at 37 °C in intracellular-like medium containing 50 µM CaCl2 (with 150 Ci/mol 45Ca2+), buffered to 0.1 µM free Ca2+ with EGTA, 3% polyethylene glycol, and 5 µM ruthenium red (to prevent mitochondrial Ca2+ accumulation) in a total volume of 2 ml. Effectors mentioned in the figures (InsP3, adenophostin-A, and 2-APB) were added at the times shown. Oxalate with GTP when present were added immediately before the start of uptake. At the required times, 200-µl aliquots were removed from the stirred uptake medium, diluted immediately into 4 ml of ice-cold intracellular-like medium containing 1 mM LaCl3, then rapidly vacuum-filtered on glass fiber filters (Whatman GF/B), washed, and counted. The figures show ATP-dependent Ca2+ accumulation with that component of Ca2+ retained by cells and filters in the absence of ATP subtracted (~0.1% of total Ca2+). All experiments shown are typical of at least three and, in most cases, a larger number of separate experiments.

Electroretinogram Recordings in Drosophila Retina-- Wild-type (Canton S), trp, and trpl mutant flies were immobilized on coverslips in bee's wax, and electroretinogram (ERG) recordings were performed as previously described using orange (580 nm) light (45). To introduce 2-APB or the control solution into the retina of a live fly, a small hole was generated near the edge of the eye using a glass needle. The hole was covered immediately with Vaseline to prevent dehydration of the exposed tissue. 2-APB (100 mM in Me2SO) or Me2SO (negative control) was diluted 1:200 into Ringer's solution, and glass needles (tip diameter ~8 µm) containing these solutions were inserted into the holes in the retina. The chemicals were driven into the eye by application of brief pressure using a 1-ml syringe connected to the needles. The injections caused an immediate change in the ERG amplitudes due to an alteration in electrical resistance. This initial change was also observed with vehicle only and therefore did not represent an effect of the 2-APB.

Materials and Miscellaneous Procedures-- InsP3 and adenophostin-A were from Calbiochem. ATP, GTP, EGTA, carbachol, polyethylene glycol, saponin, ruthenium red, Hepes, and oxalate were purchased from Sigma. 2-APB used in intact cell studies was from Sigma. For the 45Ca2+ studies using permeabilized DT40 cells, we utilized 2-APB from Tocris (Ballwin, MO) since Sigma 2-APB induced significantly greater inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump-mediated Ca2+ uptake. Thapsigargin was from LC Services, Woburn, MA. Fura-2/acetoxymethylester was from Molecular Probes, Eugene, OR. The DT40 cell line was from Dr. Tomohiro Kurosaki, Kyoto, Japan. For Ca2+ flux experiments, free Ca2+ concentrations were controlled using EGTA, computing all complexes between EGTA, ATP, Ca2+, Mg2+, monovalent cations, and protons, as previously described (46).

    RESULTS AND DISCUSSION
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ABSTRACT
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The mechanism of coupling between intracellular Ca2+ stores and the activation of store-operated Ca2+ entry channels has remained an elusive process. Support for the conformational-coupling model (9, 10) has arisen from recent evidence that InsP3 receptors are involved in the activation of TRP channels and SOCs (17-20). Availability of the membrane-permeant antagonist of the InsP3 receptor, 2-APB (47), provided some new information on the possible role of the InsP3 receptor in capacitative Ca2+ entry (17). 2-APB blocks the activation of capacitative Ca2+ entry in response to store depletion with Ca2+ pump blockers or ionomycin (17, 21). In addition, 2-APB blocks receptor-induced activation of mammalian TRPC3 channels (17, 21). TRPC3 channels can also be activated directly by application of diacylglycerol (17, 36); however, this direct stimulation of TRPC3 channels is not blocked by 2-APB, suggesting its action is not directly on the Ca2+ entry channels but, instead, on the coupling process, leading to channel activation in response to receptor stimulation (17).

Using the T3-65 clone of HEK293 cells stably expressing TRPC3 channels (48), the experiment shown in Fig. 1 appeared to reinforce this conclusion. In these cells, TRPC3 channels can be distinguished from SOCs by their ability to mediate a substantial entry of Sr2+ in response to agonists of phospholipase C-coupled receptors (17). Carbachol acting through endogenous muscarinic receptors was observed to be a particularly effective activator of TRPC3 channels. Compared with other phospholipase C-coupled receptors in the same cells, for example purinergic receptors (17), muscarinic receptor-induced InsP3 levels are sustained longer as a result of less efficient receptor desensitization (49). At a low dose (1 µM), carbachol induced a modest transient release of Ca2+ from internal stores in the absence Ca2+, and a substantial entry of Sr2+ upon the addition of the divalent cation, typical of the activation of TRPC3 channels in these cells. In the presence of 75 µM 2-APB, both the release and entry responses to 1 µM carbachol were abolished (Fig. 1B). At higher concentrations of carbachol (Fig. 1, C and E), both the release phase and the entry phase were more pronounced. However, the inhibitory action of 2-APB was substantially decreased. With 10 µM carbachol, significant release and entry were observed in the presence of 2-APB (Fig. 1D), and at 100 µM carbachol, release and entry were reduced by only ~50% by 2-APB (Fig. 1F). This could suggest that the higher levels of InsP3 induced by muscarinic activation prevent the action of 2-APB on both InsP3 receptor-mediated Ca2+ release and TRPC3 channel activation. Such an effect would be consistent with the action of 2-APB on the InsP3 receptor, which has been shown to be reduced at higher InsP3 concentrations (47). The implication from this is that the InsP3 receptor could be the mediator of both release and entry, a conclusion consistent with the substantial evidence indicating a direct interaction between TRP channels and InsP3 receptors (18-20). It is also possible that the higher activation of phospholipase C results in more diacylglycerol (as well as InsP3) to stimulate TRPC3 channels (36), resulting in the observed decrease in 2-APB sensitivity since the action of diacylglycerol on TRPC3 channels is insensitive to 2-APB (17). Whether or not this is the case, it still remains that 2-APB blocks receptor-induced TRPC3 activation.


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Fig. 1.   Inhibition by 2-APB of muscarinic receptor-induced Ca2+ release and Sr2+ entry through TRPC3 channels stably transfected HEK293 cells is partially reversed by increased agonist stimulation. Cytosolic Ca2+ was measured in fura-2-loaded T3-65 clone of the HEK293 cells as described under "Experimental Procedures." Standard conditions included 1 mM Ca2+ in the external medium; bars indicate times of replacement of nominally divalent cation-free medium with medium containing 1 mM Sr2+ and/or 75 µM 2-APB. A, TRPC3 channels were activated by the addition of 1 µM carbachol (CCh) (arrow) followed by the addition of medium containing 1 mM Sr2+. B, as in A, but in the presence of 75 µM 2-APB (bar). C, as in A, but TRPC3 channels were activated by the addition of 10 µM CCh (arrow) followed by the addition of medium containing 1 mM Sr2+. D, as in C, but in the presence of 75 µM 2-APB (bar). E, as in A, but hTRP3 channels were activated by the addition of 100 µM carbachol (arrow) followed by the addition of medium containing 1 mM Sr2+. F, as in E, but in the presence of 75 µM 2-APB (bar). F, fluorescence.

The central question of whether the action of 2-APB on TRPC3 channels reflects its modification of InsP3 receptors was addressed by analyzing the TRP channels that mediate the visual response in Drosophila. Invertebrate phototransduction occurs through rhodopsin-induced phospholipase C activation, resulting in activation of the family of retinal-specific TRP channels that mediate the light-induced current (16). The activation of these channels had been considered a paradigm for store-operated channels; however, the role of Ca2+ stores in the light response is controversial, and evidence indicates they may not be involved (16, 37). Moreover, genetic studies indicate that the single InsP3 receptor gene product in Drosophila is dispensable with respect to the light response (50, 51). Based on the action of 2-APB on the mammalian TRPC3 channels, it was important to assess the agent's action on Drosophila TRP channels. Drosophila photoreceptor cells express three members of the TRP family, TRP, TRPL, and TRPgamma (52-54). Loss-of-function mutations in two of these channels, TRP and TRPL, have been described, demonstrating that they are essential for a normal light response (55, 56). To determine whether 2-APB had any effect on the light-stimulated channel activities, the drug was injected into fly eyes, and ERG recordings were performed. ERGs are extracellular recordings that measure the summed response of all cells in the retina. Since TRP, TRPL, and TRPgamma form the light-stimulated channels in photoreceptor cells, the ERG amplitudes reflect the activities of these TRP family members. Stimulation of wild-type eyes with light results in a corneal negative response that rapidly returns to base line after cessation of the light stimulus. Control injections in wild-type retinas had no effect on the response amplitudes of the ERG over the 4-min course of the experiment (Fig. 2A, top trace). The ERGs in trp and trpl flies were also unchanged after control injections (data not shown). Injection of 2-APB caused a gradual decrease in amplitude of the wild-type ERG responses (Fig. 2A, second trace). The trp and trpl mutants were more sensitive to the inhibitory effects of 2-APB than wild-type flies (Fig. 2A, third and fourth traces). A plot of the time course of inhibition of light response amplitude by 2-APB is shown in Fig. 2B. Four minutes after injection of 2-APB, the ERG amplitudes of wild-type flies were decreased to 46.5 ± 4.1%, whereas those in trp or trpl flies were inhibited to 21.1 ± 3.7% and 18.8 ± 2.1% respectively.


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Fig. 2.   The TRP channel-mediated light responses in Drosophila eyes are inhibited by 2-APB. A, ERG recording in wild-type (WT) flies (Canton S strain) after introduction of the vehicle alone (Me2SO) diluted in Ringer's solution (ctrl inj). The flies were exposed to a series of 2-s orange light stimuli with a frequency of 2 pulses/min. An ERG recorded 4 min after the control injection is included to the right. Similar control injections were performed with trp and trpl flies (data not shown). trp flies were exposed to light stimuli of slightly shorter (1.7 s) to avoid the inactivation resulting from repeated exposure to light of 2-s duration. As was the case with wild-type flies, the amplitudes of the trp and trpl ERGs were largely unchanged over the course of the experiment (data not shown). Event markers showing the initiation and cessation of the light stimuli are indicated above, and time and millivolt scales are presented to the right of the ERGs. B, ERG recordings after the introduction of 2-APB (500 µM final concentration) to the retinas of wild-type (WT), trp, and trpl flies. WT and trpl flies were exposed to light pulses of 2-s duration, and trp flies were stimulated with 1.7-s light pulses. C, relative amplitudes of ERGs in wild-type, trp, and trpl flies after exposure to 2-APB or the control injections. Shown are the mean ERG amplitudes at a frequency of 2 light pulses/min over the course of 4 min. The numbers of flies assayed (n) are indicated. The zero time point represents the first light response 7 s after the injections.

Given the information from the laboratories of Zuker and co-workers (50) and Hardie and co-workers (51) that activation of the TRP channels mediating the Drosophila light-induced current appears normal in the absence of InsP3 receptors, the results indicate that the action of 2-APB may be independent of the InsP3 receptor. Although the action of 2-APB on the Drosophila ERGs may appear slower than its action on SOCs and TRP channels in mammalian cells (17, 21), it should be noted that the ERG is a measurement of the sum of responses from all retinal cells, only some of which are in close apposition to the site of 2-APB application. Diffusion of the drug to more distant cells likely accounts for the slower onset of its action. Indeed, it appears that almost complete inhibition of TRP channel activation can be obtained with 2-APB within the 4-min time period. The principle channels in wild-type photoreceptor cells appear to be TRP homomultimers, TRP/TRPL heteromultimers, and TRPL/TRPgamma heteromultimers (54, 57). Because TRP/TRPL heteromultimers are absent in both trp and trpl, the greater sensitivity of the mutant flies to the effects of 2-ABP may reflect a relative lower sensitivity of TRP/TRPL heteromultimers to 2-ABP. From these studies it is probable that the action of 2-APB on blocking TRP channels, at least in fly retinal cells, is through a target other than the InsP3 receptor.

Considering this information, it was important to assess whether the action of 2-APB on SOC activation in vertebrate cells was also independent of the InsP3 receptor. An important tool for such analysis was the InsP3 receptor knock-out cell line developed by Sugawara et al. (38). The wild-type DT40 chicken B cell line is a useful tool for generating gene knock-outs and expresses all three mammalian subtypes of the InsP3 receptor (38). The loci of the three InsP3 receptor subtypes were disrupted sequentially by the targeted introduction of mutations of the alleles of each InsP3 receptor subtype into the wild-type loci by homologous recombination (38). The resulting triple-InsP3 receptor gene knock-out cells (DT40InsP3R-k/o) have been reported to have no phospholipase C-coupled receptor-induced Ca2+ release responses, yet apparently retain Ca2+ entry in response to store-depletion (38). It has been suggested that the triple InsP3R-k/o cells could be expressing truncated variants of the InsP3 receptor and that certain functions of the InsP3 receptor (for example, physical coupling to plasma membrane entry channels) could be retained in the cells (18); however, there is at present no evidence that a corresponding transcript or protein is expressed in these cells.

Using both wild-type DT40 cells and the DT40 triple InsP3R-k/o cells, we sought first to assess and compare InsP3 receptor function directly. Radioactive Ca2+ flux measurements using permeabilized cells provide a sensitive means for assessing the Ca2+ accumulation and release properties of Ca2+ stores (43, 46) and have not previously been conducted in the InsP3R-k/o cells. Saponin-permeabilized DT40w/t cells displayed sarcoplasmic/endoplasmic reticulum Ca2+ ATPase-mediated Ca2+ pumping activity qualitatively similar to many other cell types (42-44), although the absolute uptake capacity of stores (between 0.1 and 0.2 nmol of Ca2+/106 cells) was somewhat less, likely as a result of the comparatively smaller size of DT40 cells. In DT40w/t cells, application of 10 µM InsP3 induced rapid release of more than 50% of accumulated Ca2+ (Fig. 3A). Ca2+ accumulation was almost identical in the DT40InsP3R-k/o cells; however, in contrast to DT40w/t cells, there was no measurable release of Ca2+ in response to InsP3 (Fig. 3B). Moreover, adenophostin A, a powerful InsP3 receptor agonist with 100-fold greater potency than InsP3 (58), also had no significant effect on Ca2+ release in the DT40InsP3R-k/o cells (Fig. 3C).


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Fig. 3.   InsP3- and adenophostin A-mediated effects on Ca2+ release in permeabilized wild-type and triple InsP3 receptor knockout variants of the DT40 cell line. Cell permeabilization and ATP-dependent Ca2+ accumulation were as described under "Experimental Procedures." A, uptake was under control conditions () or with 10 µM InsP3 (open circle ) added at 4 min (arrow) in wild-type DT40 cells (DT40wt). B, as in A, but cells were the triple InsP3 receptor knockout variant of the DT40 line (DT40InsP3R-k/o). C, as with B, but with 500 nM adenophostin A (Adn-A) (open circle ) added at 4 min (arrow).

The somewhat small accumulation of Ca2+ within DT40 cells made it difficult to conclude with certainty that there was no InsP3 receptor function. Uptake of Ca2+ within the ER lumen can be dramatically enhanced by application of a combination of GTP and oxalate to the uptake medium (41, 42, 59). GTP induces self-association and communication between ER subcompartments within permeabilized cells, resulting in luminal Ca2+ accumulation reflecting the combined activity of a much large number of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pumps (42, 46, 59). The InsP3-sensitive Ca2+ pool is selectively permeable to carboxylate anions, including oxalate, which precipitate Ca2+ and greatly increase the Ca2+ capacity of the ER lumen (41, 42, 46). Whereas 10 mM oxalate alone has a marginal effect, in combination with 20 µM GTP there is an ~100-fold increase in the accumulation of Ca2+ specifically within the InsP3-sensitive Ca2+ pool (59, 60). In this respect, DT40 cells were similar, and Ca2+ accumulation in the presence of GTP and oxalate continued at a steady rate (Fig. 4). This rate of uptake was sustained for 10s of minutes, resulting in accumulation of Ca2+ 20-50-fold higher than the steady state reached in the absence of GTP and oxalate. Almost all of this Ca2+ uptake was within the InsP3-sensitive Ca2+ pool. Thus, inclusion of InsP3 in the uptake medium resulted in a substantial reduction of Ca2+ accumulation (Fig. 4A). From a number of experiments the effect of InsP3 was to prevent 80-90% of the Ca2+ accumulation in DT40w/t cells (Fig. 4A). In complete contrast, there was no effect of InsP3 on Ca2+ accumulation in DT40InsP3R-k/o cells even though there was an almost identical enhancement of Ca2+ accumulation in the presence of GTP and oxalate (Fig. 4B). Even more compelling were the differences in effectiveness of adenophostin A under the same conditions (Fig. 4, C and D); almost 100% of the accumulation was prevented in DT40w/t cells, whereas there was no releasing action in the DT40InsP3R-k/o cells. The slight enhancement of Ca2+ accumulation observed with adenophostin A in Fig. 4D was not consistent; in four separate experiments with knock-out cells, there was no measurable decrease in uptake with adenophostin A. Thus, under conditions enormously enhancing the amount of Ca2+ sequestered within the InsP3-sensitive Ca2+ store and, hence, the Ca2+ release signal mediated by InsP3 receptors, the results clearly reveal the absence of any functional InsP3 receptors in the knock-out cells, whereas the responses of wild-type cells are essentially normal. An interesting corollary to this is that, in answer to an earlier question (11), the action of GTP to induce communication between ER subcompartments does not appear related to InsP3 receptor function.


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Fig. 4.   Effects of InsP3 and adenophostin-A on Ca2+ sequestration enhanced by GTP and oxalate in permeabilized wild-type DT40 cells and permeabilized cells of the InsP3-receptor knockout variant of the DT40 cell line. ATP-dependent Ca2+ accumulation was measured in the presence of 20 µM GTP and 10 mM oxalate. All additions were made before initiation of Ca2+ accumulation by ATP addition. The conditions were as follows: A, control conditions with 20 µM GTP and 10 mM oxalate () or with 10 µM InsP3 (open circle ) in the wild-type DT40 cells (DT-40wt); B, as in A, but using the InsP3 receptor knockout variant of the DT40 cells (DT40InsP3R-k/o); C, as in A, but with 500 nM adenophostin A (Adn-A) (open circle ); D, as in B, but with 500 nM Adn-A ().

Given this remarkable divergence in store function measured in the permeabilized cells, it was crucial to assess whether there were any corresponding changes in the operation of stores and the activation of store-operated Ca2+ entry in the intact cells. The data in Fig. 5, A and B, reveal that store function and store-operated Ca2+ entry were each virtually identical in the intact DT40w/t and DT40InsP3R-k/o cells. The release of Ca2+ in response to thapsigargin was the same in both amplitude and rate, indicating that the size of stores and the leak rate from stores after pump blockade were unaffected by the complete absence of functional InsP3 receptors. This is an interesting result in itself since it indicates that leak of Ca2+ is not attributable to the function of InsP3 receptors. After removal of extracellular Ca2+, the decrease in cytosolic Ca2+ indicated that the component of the response resulting from store-operated Ca2+ entry was clearly the same in the two cell lines. Readdition of Ca2+ resulted in the almost identical resumption of store-operated Ca2+ entry, confirming the earlier work of Sugawara et al. (38). In many cell types, removal and readdition of Ca2+ after store-emptying results in a large overshoot of store-operated Ca2+ entry, reflecting transient reversal of a Ca2+-sensitive negative feedback mechanism on SOC activation (11, 61). In contrast, the entry response upon the readdition of Ca2+ in both DT40 cell lines did not include an overshoot, entry merely returning to an approximate steady state level.


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Fig. 5.   Ca2+ store mobilization induced by thapsigargin and subsequent store-operated Ca2+ entry in intact DT40 wild-type cells and the DT40 triple InsP3 receptor knockout variant cell line. Ca2+ measurements were as described under "Experimental Procedures"; bars indicate times of replacement of medium with nominally Ca2+-free media (0 Ca2+). A, Ca2+ levels were monitored after store depletion with 1 µM thapsigargin (arrow) and transient Ca2+ removal in the wild-type DT40 cells (DT40wt). B, as in A, but in the InsP3 receptor knockout DT40 variant cells (DT40InsP3R-k/o). F, fluorescence.

Since SOC activation occurred essentially normally in the two cell types, the important question was whether the entry was sensitive to the InsP3 receptor antagonist, 2-APB. The answer to this question is clearly affirmative, as shown from the data in Fig. 6. In this experiment, Ca2+ entry was activated by Ca2+ addition after thapsigargin-induced store depletion in the absence of extracellular Ca2+. A comparison of release and entry for DT40w/t and DT40InsP3R-k/o cells under this condition is shown in Fig. 6, A and B, respectively. If 75 µM 2-APB was added 5 min before Ca2+ addition, the store-operated Ca2+ entry was almost completely blocked in both cell types (Fig. 6, C and D). As important as determining whether 2-APB could alter SOC activity was to determine if the presence or absence of functional InsP3 receptors might influence the kinetics of action of 2-APB on SOC function. The data shown in Fig. 7 address this point. After emptying stores in DT40-w/t cells with thapsigargin, the addition of Ca2+ resulted in sustained Ca2+ entry lasting for 10s of minutes. The addition of 2-APB caused an immediate inhibition of SOC-mediated Ca2+ entry, and Ca2+ levels rapidly fell (Fig. 7A). This action of 2-APB was identical in the DT40InsP3R-k/o cells (Fig. 7B). Moreover, the presence or absence of InsP3 receptors did not influence the return of functional SOC activity after removal of 2-APB. Thus, after the blockade of SOC-mediated Ca2+ entry, removal of 2-APB caused a clear return of Ca2+ entry in DT40w/t cells (Fig. 7C), and this reversal of action of 2-APB was almost identical in DT40InsP3R-k/o cells (Fig. 7D). Experiments also assessed the concentration dependence of action of 2-APB to block store-operated Ca2+ entry in the two cell types. There was no significant difference in the 2-APB sensitivity of Ca2+ entry between DT40w/t and DT40InsP3R-k/o cells, the IC50 in both cases being 15-20 µM (data not shown). Therefore, the actions of 2-APB on store-operated Ca2+ entry does appear to be independent of whether or not cells have functional InsP3 receptors.


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Fig. 6.   Store-operated Ca2+ entry is blocked by 2-APB in both wild-type DT40 cells and the InsP3 receptor knockout variant DT40 cell line. Ca2+ measurements using the wild-type and InsP3 receptor knockout variant of the DT-40 cell line were as described in the legend to Fig. 5; bars indicate times of replacement of medium with nominally Ca2+-free media (0 Ca2+) and/or 75 µM 2-APB. A, Ca2+ release induced by the addition of 1 µM thapsigargin (arrow) in Ca2+-free media followed by Ca2+ entry after the addition of medium containing 1 mM Ca2+ in the wild-type DT-40 cells (DT40wt). B, as in A, but in the InsP3 receptor knockout variant of the DT40 cells (DT40InsP3R-k/o). C, as in A, but in the presence of 75 µM 2-APB (bar) added before the addition of medium containing 1 mM Ca2+. D, as in B, but in the presence of 75 µM 2-APB (bar) added before the addition of medium containing 1 mM Ca2+. F, fluorescence.


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Fig. 7.   Acute addition of 2-APB rapidly blocks store-operated Ca2+ entry after store depletion, and this inhibition by 2-APB is reversible in both the wild-type and the InsP3-receptor knockout variant of the DT-40 cell line. Ca2+ measurements using the wild-type and InsP3 receptor knockout variant of the DT-40 cell line were as described in the legend to Fig. 5; bars indicate times of replacement of medium with nominally Ca2+ free media (0 Ca2+) and/or 75 µM 2-APB. A, Ca2+ entry upon the addition of medium containing 1 mM Ca2+ in wild-type DT40 cells (DT40wt) after prior pool depletion with 1 µM thapsigargin (not shown) can be blocked by the acute addition of 75 µM 2-APB (bar). B, as in A, but in the InsP3 receptor knockout variant of the DT40 cell line (DT40InsP3R-k/o). C, recovery of store-operated Ca2+ entry after the removal of 75 µM 2-APB in wild-type DT40 cells (DT-40wt) after pool depletion with 1 µM thapsigargin (arrow). D, as in C, but in the InsP3 receptor knockout variant of the DT40 cells (DT40InsP3R-k/o). F, fluorescence.

Considering these results, one further important question about 2-APB was whether it truly did have a direct action upon the InsP3 receptor within DT40 cells. Until now we have used intact cells and shown that 2-APB clearly blocks phospholipase C-coupled receptor agonist responses in a variety of cell types, and this has been presumed to reflect blockade of InsP3-induced activation of InsP3 receptors (17, 21). However, since the actions of 2-APB on Ca2+ entry in DT40 cells appear independent of InsP3 receptors, we considered the possibility that the InsP3 receptor may not be a direct target of 2-APB in these or even other cells. It was important to investigate the direct action of 2-APB on the InsP3 receptors in permeabilized DT40w/t cells. As shown in Fig. 8, 2-APB was a highly effective functional blocker of InsP3 receptors in DT40w/t cells. Thus, under standard conditions in which InsP3 induced rapid Ca2+ release (Fig. 8A), the prior addition of 50 µM 2-APB completely prevented the action of InsP3 (Fig. 8B). Moreover, under conditions in which maximal sustained Ca2+ accumulation within the InsP3-sensitive Ca2+ pool was achieved in the presence of GTP and oxalate, the action of InsP3 to effect release and prevent Ca2+ accumulation (Fig. 8C) was completely blocked by 2-APB (Fig. 8D). In these experiments 2-APB had a modest inhibitory effect on Ca2+ pumping activity in DT40w/t cells; this was the same for pumping activity in DT40InsP3R-k/o cells (data not shown), indicating that this was not a reflection of an action of 2-APB on the InsP3 receptor. Last, we undertook experiments to determine the sensitivity of action of 2-APB on the InsP3 receptor in permeabilized DT40w/t cells (Fig. 9). The IC50 for 2-APB on InsP3-mediated Ca2+ release was ~12 µM (Fig. 9, inset).2 Although not identical, this value is close to the IC50 values for 2-APB on inhibition of store-operated Ca2+ entry in intact DT40 cells, both wild-type and knock-outs, which as described above were both 15-20 µM. Moreover, these values are very similar to the IC50 values for 2-APB on store-operated channels and TRPC3 channels, which were measured as 10 µM and 10-15 µM, respectively, in HEK293 cells (17).


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Fig. 8.   InsP3-induced Ca2+ store release is completely blocked by 2-APB in permeabilized wild-type DT-40 cells. ATP-dependent Ca2+ accumulation and oxalate-induced GTP-dependent Ca2+ sequestration in permeabilized wild-type DT40 cells were measured as described in the legends to Figs. 3 and 4. A, uptake was under control conditions () or with 10 µM InsP3 (open circle ) added at 4 min (arrow). B, as in A, but with 50 µM 2-APB present throughout (bar). C, ATP-dependent Ca2+ accumulation was measured under control conditions in the presence of 20 µM GTP and 10 mM oxalate () or in the presence of 10 µM InsP3 (open circle ). All additions were made before Ca2+ accumulation. D, as in C, but with 50 µM 2-APB present throughout (bar).


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Fig. 9.   Concentration dependence of 2-APB-induced inhibition of Ca2+ release through InsP3 receptors in permeabilized wild-type DT40 cells. ATP-dependent Ca2+ accumulation and oxalate-induced GTP-dependent Ca2+ sequestration in permeabilized wild-type DT40 cells were measured as described in the legends to Fig. 3. Uptake was under control conditions () or with 10 µM InsP3 and either without 2-APB (open circle ) or with 5 µM 2-APB (black-triangle), 15 µM 2-APB (triangle ), 35 µM 2-APB (), or 50 µM 2-APB (black-square) present throughout. All additions were made prior to Ca2+ accumulation. Inset, plot of the concentration dependence of the action of 2-APB on InsP3 receptor-mediated Ca2+ release in permeabilized DT40 cells, revealing an IC50 of ~12 µM.

Overall, these studies provide significant new information on the activation of TRP channels and store-operated Ca2+ entry channels. The actions of 2-APB in the InsP3 receptor knock-out cells provide compelling evidence that the target through which 2-APB modifies store-operated Ca2+ entry is unlikely to be any of the known InsP3 receptor gene products. Certainly, our data provide strong evidence that the DT40-InsP3R-k/o cells are devoid of any functional InsP3 receptors. The only uncertainty about this conclusion is the possibility that the inserted mutated InsP3 receptor gene sequences could give rise to expression of truncated InsP3 receptors deficient in part of the C-terminal pore-forming domain, as recently suggested (18). In experiments of Kiselyov et al. (18), a 154-amino acid C-terminal deletion construct of the type I InsP3 receptor, when expressed in HEK293 cells, was shown to couple and allow InsP3-mediated activation of co-expressed TRPC3 channels. Thus, whereas the C-terminal transmembrane domains of the InsP3 receptor form the release channel, the large cytoplasmic domain appears to couple directly with plasma membrane entry channels (19, 20). However, although the expression of C-terminal deletions could provide an InsP3 receptor target for 2-APB and account for inhibition of SOC activation in the DT40-InsP3R-k/o cells, at present there is no evidence for the existence of any transcript or translation product corresponding to a fragment of the InsP3 receptor in these cells.3

The conclusion that a target other than the InsP3 receptor mediates the action of 2-APB on entry channels gains further support from the ability of 2-APB to inhibit TRP channel activation in Drosophila phototransduction. In Drosophila, there is only a single InsP3 receptor subtype, and homozygous elimination of the InsP3 receptor in fly eyes in the experiments of Acharya et al. (50) and Raghu et al. (51) establish that this InsP3 receptor is not required for rhodopsin-mediated activation of the wild-type light-induced current response. Therefore, the action of 2-APB to inhibit the visual TRP channels may be either by direct channel inhibition or it may reflect interaction of 2-APB with another regulatory protein component that is not the InsP3 receptor. Our results indicate that the action of 2-APB is not restricted to a particular member of the family of TRP channels mediating phototransduction. Thus, it would seem that activation of at least two and possibly all three Drosophila TRP channels is blocked by 2-APB.

However, recent work suggests that 2-APB may not necessarily have a direct inhibitory action on the TRP channels themselves. Thus the Drosophila retinal TRP channels can be reversibly activated by induction of metabolic stress, for example anoxia, ATP depletion, or mitochondrial uncoupling (62). This activation occurs independently of light stimulation and does not require rhodopsin, Gq, or phospholipase C, suggesting it is at a late coupling stage downstream of the light-induced pathway and perhaps upon the channel directly. However, whereas light induction of TRP channels is blocked by 2-APB, their direct activation in response to metabolic stress is not blocked by 2-APB.4 This situation is intriguingly analogous to the actions of 2-APB on mammalian TRPC3 channels. Thus, activation of TRPC3 channels by physiological phospholipase C-coupled receptors is blocked by 2-APB, but direct channel activation by application of diacylglycerol is resistant to 2-APB (17). These results would lead us to conclude that the target for 2-APB mediates a step that is upstream from the channel in the pathway leading to physiological activation of TRP channels. Whereas we cannot rule out the possibility that 2-APB is a direct modifier of Ca2+ entry channels, we should consider that its actions are rather specific. Thus, 2-APB has little effect on a range of Ca2+ and related channels including ryanodine receptors (47), voltage-sensitive Ca2+ entry channels (47), arachidonic acid-activated Ca2+ entry channels (63), Ca2+ entry channels activated by S-nitrosylation (21), Ca2+-activated Cl- entry channels (64),4 or purinergic P2X receptor Ca2+ entry channels.5 Instead, its action appears restricted to preventing activation of TRP channels, SOCs, and, of course, InsP3 receptors.

Recent data from Braun et al. (65) indicates that 2-APB can block a Ca2+ channel activity measured in excised patches from rat basophilic leukemia cells. However, it is not clear whether this current reflects authentic store-operated channels mediating the Ca2+ release-activated current (ICRAC). The suggestion was that the action of 2-APB was directly at the plasma membrane. One could argue that since 2-APB may be a direct modifier of InsP3 receptors (especially considering the data presented in Fig. 8), then it could as easily be a direct modifier of TRPs and SOCs. However, this would predict that TRPs, SOCs, and InsP3 receptors would share some underlying functional/structural motifs. Considering all these arguments, perhaps a reasonable hypothesis for the action of 2-APB is that it interacts with a target, possibly a regulatory protein, which directly mediates control over SOCs, TRP channels, and InsP3 receptors. Certainly, the important conclusions to be drawn from this work are that, although InsP3Rs do not mediate the action of 2-APB on Ca2+ entry, the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

    ACKNOWLEDGEMENTS

We thank Dr. Lutz Birnbaumer for providing the transfected HEK293 cells and Dr. Katsuhiko Mikoshiba for helpful advice.

    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grant HL55426 (to D. L. G.), a fellowship from the American Heart Association, Maryland Affiliate (to H-T. M.), a fellowship from the Interdisciplinary Muscle Training Program (to R. L. P.), and NIH Grant EY10852 (to C. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger The contribution of these two authors was equal.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Tel.: 410-706-2593 (office) and 410-706-7247 (laboratory); Fax: 410-706-6676; E-mail: dgill@umaryland.edu.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100944200

2 In work published by Missiaen et al. (66) subsequent to the submission of this paper, the IC50 for 2-APB on InsP3 receptor-induced Ca2+ release in permeabilized A7r5 cells was measured as 36 µM. The difference may be cell type-related or may reflect differences between the sources of 2-APB as described under "Materials and Miscellaneous Procedures."

3 T. Kurosaki, unpublished observations.

4 I. Chorna-Ornan, T. Joel-Almagor, H. C. Ben-Ami, S. Frechter, B. Gillo, Z. Selinger, D. L. Gill, and B. Minke, submitted for publication.

5 S-D. Mu, H-T. Ma, K. Venkatachalam, and D. L. Gill, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; TRP, transient receptor potential, InsP3, inositol 1,4,5-trisphosphate; fura-2/AM, fura-2 acetoxymethylester; 2-APB, 2-aminoethoxydiphenyl borate; SOC, store-operated channel; ERG, electroretinogram; HEK cells, human embryonic kidney cells.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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