The acidocalcisome inositol-1,4,5-trisphosphate receptor of Trypanosoma brucei is stimulated by luminal polyphosphate hydrolysis products

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei. However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40–3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of acidocalcisomes in T. brucei.

Inositol-1,4,5-trisphosphate receptors (IP 3 Rs) 2 are intracellular Ca 2ϩ channels mostly found in the endoplasmic reticulum (ER) of animal cells (1). When plasma membrane receptors are stimulated, activation of a phospholipase C results in IP 3 formation and opening of these channels leading to a rise in cytosolic Ca 2ϩ (2). IP 3 and Ca 2ϩ function as co-agonists of IP 3 Rs, and Ca 2ϩ release from an IP 3 R leads to opening of its neighbors and, consequently, IP 3 -regulated Ca 2ϩ -induced Ca 2ϩ release (CICR) with the eventual generation of Ca 2ϩ waves (3). The IP 3 R has been considered essential for Ca 2ϩ signaling in animals and for the regulation of a variety of processes including gene expression, signal initiation, contraction, secretion, proliferation, fertilization, development, and cell death (4). Constitutive IP 3 -mediated Ca 2ϩ transfer to mitochondria is essential for maintaining cellular bioenergetics (5).
Trypanosomes, like Trypanosoma brucei, which causes African trypanosomiasis or sleeping sickness, and Trypanosoma cruzi, the agent of Chagas' disease, belong to the eukaryotic supergroup Excavata (6) and possess a very peculiar acidic calcium store, the acidocalcisome (7,8). Acidocalcisomes are rich in polyphosphate (polyP), a polymer of orthophosphate linked by high-energy phosphoanhydride bonds, and found in a diverse range of organisms (9). We have shown that the IP 3 R of T. brucei is localized to acidocalcisomes rather than to the ER and is maximally activated at high IP 3 concentrations (10 -20 M) (10,11). The acidocalcisome localization was also confirmed in T. cruzi (12). We also demonstrated that Ca 2ϩ signaling through the TbIP 3 R has roles in parasite growth in vitro and in vivo (10). The relevance and essentiality of the IP 3 R for growth in vitro and in vivo and for differentiation were also demonstrated in T. cruzi (13).
Several proteins are known to interact with different IP 3 R mammalian isoforms and to modify their activity (4). Most of them interact with the cytosolic portion of the IP 3 R. However, some proteins, like the Ca 2ϩ storage proteins chromogranin A and B, have been shown to interact with the luminal portion of the IP 3 R (4). Acidocalcisomes apparently have few luminal proteins but they do have polyP, which has been shown to modulate the function of ion channels (20).
To characterize the TbIP 3 R we used chicken B lymphocytes in which the genes for all three vertebrate IP 3 Rs have been stably eliminated (DT40 -3KO) (21). We previously stably trans-This work was supported by NIAID, National Institutes of Health Grants AI-108222 (to R. D.) and T32 AI060546 (to E. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1 To whom correspondence should be addressed. cro ARTICLE fected these cells with the gene encoding TbIP 3 R or the rat IP 3 R1 (RnIP 3 R1), used as positive control (10). Heterologous expression of the TbIP 3 R and RnIP 3 R1 was confirmed by immunofluorescence analysis, which showed ER localization, and by Western blot analysis (10). Here, we report that opening of the TbIP 3 R expressed in DT40 -3KO cells is stimulated by the luminal polyP hydrolysis products, P i and PP i or by alkalinization.

TbIP 3 R expression in DT40 cells and regulation by Ca 2؉ and ATP
In mammalian cells, low intracellular free calcium ([Ca 2ϩ ] i ) potentiates whereas high [Ca 2ϩ ] i inhibits channel activity. The mechanism of this regulation is not yet clear (17), but it has been shown that a conserved glutamate residue (Glu-2100) in the regulatory domain of mammalian IP 3 R is important to determine its Ca 2ϩ sensitivity (22). TbIP 3 R contains a conserved glutamate residue homologous to the mammalian Glu-2100 (Glu-2405) (Fig. 1). To investigate whether TbIP 3 R is regulated by Ca 2ϩ , we trapped a low-affinity Ca 2ϩ indicator (Mag-Fluo4) within the ER to measure luminal free [Ca 2ϩ ] in saponin-permeabilized DT40 -3KO-TbIP 3 R cells. Addition of MgATP stimulated Ca 2ϩ uptake until a steady-state Ca 2ϩ loading was reached (Fig. 2, A and B). Ca 2ϩ release was induced by addition of IP 3 in the presence of either 50 nM or 1 M Ca 2ϩ and either low (10 M) or high (100 M) IP 3 . Fig. 2C shows that the rate of TbIP 3 R-mediated Ca 2ϩ release is higher at 1 M Ca 2ϩ than at 50 nM Ca 2ϩ at either low (1 M) or high (100 M) IP 3 , which is consistent with previous findings using DT40 -3KO cells expressing RnIP3R1 (17). Fig. 2C shows the quantification of the changes observed in four experiments.
Animal IP 3 Rs are regulated by ATP, which binds to a glycinerich motif (GXGXXG), also known as the Walker motif (1,23,24). RnIP 3 R1 contains two Walker motifs, named ATPA and ATPB, whereas TbIP 3 R does not contain sequences corresponding to either ATPB or ATBC in the regulatory domain although it contains a Walker motif (GGLGNEGL) at the N-terminal region of the protein (suppressor domain) with similarity to the ATPA motif (GGLGLLGL) of RnIP 3 R1 (Fig. 1). Accordingly, although Ca 2ϩ loading affects the response to IP 3 (Fig. 3, A and B) the rate of Ca 2ϩ release was not significantly increased by increasing the ATP concentration from 0.3 to 3 mM (Fig. 3C). In this regard, physiological concentrations of ATP in trypanosomes are in the range of 1-3 mM (25,26). The rate of Ca 2ϩ release by IP 3 was inhibited by previous addition of the IP 3 R inhibitors 2-aminoethoxydiphenyl borate (2-APB) and heparin and (Fig. 3D).

Electrophysiological characterization of the TbIP 3 R
To characterize the electrophysiological properties of the TbIP 3 R we used nuclear patch clamp recordings. This is because the nuclear envelope is continuous with the ER membrane and channels that are normally expressed within ER membranes can pass into the outer nuclear envelope, permitting the recording of their activity from patches of nuclear membrane in a near-physiological situation (27).
Nuclei were isolated from DT40 -3KO cells stably expressing TbIP 3 R or RnIP 3 R1 as described under "Experimental procedures," and recordings were obtained from excised nuclear patches using symmetrical 140 mM K ϩ as the charge carrier, rather than Ca 2ϩ , to increase the single channel conductance (␥) and prevent feedback regulation by permeating cations. The holding potential was maintained at ϩ40 mV unless stated otherwise. Recordings were only continued if channel activity was stable. Under these conditions, channel current amplitude following pulses to negative and positive potentials were voltagedependent. Fig. 4A shows a representative example of channel activity recorded with 10 M IP 3 in the patch pipette following pulses from Ϫ60 mV to ϩ60 mV in DT40 -3KO expressing TbIP 3 R. In many patches (ϳ50%) we observed the presence of two or more active channels which may indicate TbIP 3 R clustering (28,29). Typically, channel activity was sustained over prolonged periods of time. Fig. 4B shows the current versus voltage (I-V) relationship for this channel generated from the peak K ϩ conductance at each potential in Fig. 4A. The I-V relationship behaved as a linear conductance and thus in symmetrical 140 mM K ϩ , the I-V relationship was nearly linear with an extrapolated reversal potential at 0 mV (Fig. 4B). The conductance of TbIP 3 R was typically in the range of 150 -350 pS with dominant conductance of 200 -220 pS, indicating the presence of different states (30,31). We chose IP 3 channels with one stable overtime conductance to study how different conditions could modulate Ca 2ϩ release. In our experiments, no currents were detected in the nuclear envelope from DT40 -TbIP 3 R cells when IP 3 was omitted from the patch pipette (n Ͼ100, meaning in more than 100 successful patches with gigaseal formation) (Fig. 4C). Acidic ( Fig. 4D; n Ͼ100), but not alkaline (Fig.  4E), pH also abolished the currents generated by IP 3 . Data analysis of the currents detected at different pH levels show significant changes in frequency and open probability, as well as in total power, at acidic pH, as compared with neutral (pH 7.4) or alkaline (pH 8.0) pH (Fig. 4F). In agreement with these results, alkalinization of acidic compartments by NH 4 Cl resulted in rapid Ca 2ϩ release, which was completed by addition of ionomycin ( Fig. 4, G and H). Ionomycin is not effective in releasing Ca 2ϩ from acidic compartments (32) but alkalinization of the acidocalcisomes by addition of NH 4 Cl allowed its release (8). Alkalinization of acidocalcisomes by the combination of nigericin-ionomycin also rapidly released Ca 2ϩ (Fig. 4, I and J).
The high-affinity agonist adenophostin A (in place of IP 3 ) stimulated the TbIP 3 R activity at 1 M (Fig. 5A), as previously investigated by fluorescence determination of Ca 2ϩ release in permeabilized DT40 -3KO cells expressing TbIP 3 R (10). The competitive antagonist heparin (400 g/ml, n Ͼ100) abolished ( Fig. 5B), whereas 2-APB had only partial inhibitory effect on, TbIP 3 R conductance (100 M; Figs. 5C and 3H) (33), and high concentrations of the membrane-permeable caffeine (70 mM in the bath solution; Fig. 5D) had a potent inhibitory effect on the channel conductance generated by IP 3 addition.

Effect of luminal polyP and its hydrolysis products
Acidocalcisomes are rich in P i , PP i , and short-and long-chain polyphosphate (polyP) (9). Interestingly, polyP has been shown to activate transient receptor potential (TRP) channels of the IP 3 receptor stimulated by orthophosphate and pyrophosphate melastatin family (TRPM8) (20). We therefore investigated the effect of luminal polyP and its hydrolysis products, P i and PP i , in nuclear patches of DT40 -3KO cells expressing TbIP 3 R or RnIP 3 R1. Fig. 6 shows representative current recordings obtained from excised nuclear patches of DT40 -3KO cells expressing TbIP 3 R at a holding potential of ϩ20 mV. When either P i (Fig. 6A) or PP i (Fig. 6C) was added to the bath solution (corresponding to IP 3 receptor stimulated by orthophosphate and pyrophosphate the luminal phase of acidocalcisomes) there was a significant increase in the channel conductance, as confirmed by the data analyses of four independent experiments shown in Fig. 6, B and D, respectively. In contrast, addition of polyP 3 decreased the frequency and the total power/min (Fig. 6, E and F), whereas addition of polyP 100 had no significant effect on TbIP 3 R conductance (Fig. 7, A and B).
In contrast to these results, when the current recordings of nuclear patches of DT40 -3KO cells expressing RnIP 3 R were analyzed (Figs. 7, C and D, and 8), neither P i , nor PP i , polyP 3 or polyP 100 had any effect on this channel conductance.

Discussion
Here we report the expression and electrophysiological properties of the T. brucei IP 3 R expressed in DT40 cells devoid of the three vertebrate IP 3 Rs (DT40 -3KO). TbIP 3 R has similarities and differences with vertebrate IP 3 Rs. As occurs with the vertebrate orthologs, TbIP 3 R has a very conserved C-terminal, and pore (GGGVGD) regions and has a glutamate residue (calcium sensor) in the regulatory domain whose mutation in mammalian IP 3 R alters Ca 2ϩ sensitivity (22) (Fig. 1). Accordingly, TbIP 3 R is stimulated by Ca 2ϩ . It is also inhibited by heparin and caffeine and partially by 2-APB and stimulated by

IP 3 receptor stimulated by orthophosphate and pyrophosphate
adenophostin A. In contrast to the vertebrate orthologs TbIP 3 R localizes to the acidocalcisomes (10, 11) instead of to the ER, and it has only 5 of the 10 basic residues that were proposed to form a binding pocket to accommodate the negatively charged IP 3 (34) (Fig. 1A); it does not have ATP-binding domains in the regulatory domain but instead has such a domain in the N-terminal region of the protein and is stimulated by the luminal polyP hydrolysis products, P i and PP i , and inhibited by acidic pH.
Numerous regulatory proteins interact with the cytosolic portion of the mammalian IP 3 Rs but only a few have been described to interact with their luminal portions (4). Among them the Ca 2ϩ storage proteins chromogranin A (CGA) and B (CGB), which interact with all three IP 3 R types (35)(36)(37)(38), and the ER protein 44 (Erp44) (39), which interacts with the IP 3 R type I. Chromogranins, which are located in secretory granules, increase IP 3 R activity at acidic pH (40,41), whereas Erp44 inhibits IP 3 R opening (39).
X-ray microanalysis of acidocalcisomes of different species has revealed very low or no sulfur content in their lumen, suggesting the absence of proteins or a very low protein content (9). However, the luminal region has very high concentrations of polyP, which have been calculated to reach molar levels (9). Interestingly, early 31 P NMR studies of isolated acidocalcisomes from T. brucei (42) found that the average chain length of polyP was 3.39, and we observed that polyP 3 has an inhibitory effect on TbIP 3 R conductance. The results suggest that this conductance is stimulated when polyP is hydrolyzed to P i and PP i . Acidocalcisomes of T. brucei possess a vacuolar soluble pyrophosphatase (TbVSP) that is able to hydrolyze polyP in the presence of Zn 2ϩ at an optimal pH of 6.5, more alkaline than that of acidocalcisome pH of 5.0 -5.5 (43). Alkalinization of the acidocalcisome would result in higher activity of the TbIP 3 R channel and rapid Ca 2ϩ release. In addition, it has also been reported (44) that alkalinization of acidocalcisomes results in polyP hydrolysis with accumulation of P i and PP i , which also activate the channel and would explain our Ca 2ϩ release results.
The stimulation of the acidocalcisomal TbIP 3 R by alkalinization and polyP hydrolysis products has physiological relevance when trypanosomes are submitted to osmotic stress. Under the very acidic conditions of resting acidocalcisomes (9) polyP is in its polymerized state and the channel is closed. When trypanosomes are submitted to hypo-osmotic stress it has been reported that, as a result of amino acid catabolism, there is an increase in ammonia (NH 3 ) that has been proposed to be sequestered as ammonium (NH 4 ϩ ) in acidocalcisomes, leading to their alkalinization (45). Alkalinization would lead to activation of the vacuolar soluble pyrophosphatase (43) with generation of polyP hydrolysis products leading to channel opening and Ca 2ϩ release. PolyP and Ca 2ϩ signaling have been shown to be important for the regulatory volume recovery that follows (46,47). In this regard, trypanosomes are exposed to drastic changes in osmolarity when circulating in the blood of their mammalian hosts. They must be able to resist up to 1400 mOsm when passing through the renal medulla and then return to the much lower osmotic environment of the general circulation (48).
In conclusion, by analysis of the TbIP 3 R localized in the nucleus of DT40 cells, we demonstrate that the channel is predominantly closed at acidic pH but that alkalinization or increase in P i and PP i levels in its luminal site opens it up. Therefore, the single-channel properties of TbIP 3 R are well-adapted to its role as the Ca 2ϩ release channel of acidocalcisomes.

Functional assays of IP 3 Rs in DT40 cells
Uptake of Ca 2ϩ into intracellular stores of permeabilized DT40 -3KO-TbIP 3 R or DT40 -3KO-RnIP 3 R1 cells and its release regulated by IP 3 , Ca 2ϩ , or ATP were measured using a low-affinity Ca 2ϩ indicator (Mag-Fluo4) trapped within the ER as described previously (10). Confluent cells (50 ml, 2 ϫ 10 6 cells/ml) were collected by centrifugation at 600 ϫ g for 2 min and suspended in 3 ml Hepes-buffered saline containing 1 mg/ml BSA, 0.02% (w/v) Pluronic F127, and 20 M Mag-Fluo4 AM as described (51). After incubation at 20°C for 1 h in the dark with gentle shaking, cells were centrifuged and re-suspended in 10 ml Ca 2ϩ -free cytosol-like medium (CLM) (51) containing (in mM) 140 KCl, 20 NaCl, 1 EGTA, 2 MgCl 2 , and 20 Pipes, pH 7.0, and 50 g/ml saponin (Sigma). Cells were incubated with shaking at 37°C for 4 min. Twenty-l cells were sampled to confirm permeabilization of cells by using 0.1% Trypan Blue and the cells were centrifuged and gently resuspended in 2.

DT40 nuclei isolation
We used a combination of osmotic and mechanical lysis for nuclei isolation (52). DT40 cells were centrifuged (600 ϫ g for 2 min, 4°C) and washed once with ice-cold phosphate buffer (PBS) and nuclear isolation media (NIM). PBS composition was (in mM): 137 NaCl, 2.7 KCl, 10 Na 2 PO 4, 10 KH 2 PO 4 , pH 7.4, with NaOH. NIM composition was (in mM): 250 sucrose, 150 KCl, 3 mM ␤-mercaptoethanol , 10 Tris-HCl, 1 phenylmethanesulfonyl fluoride (PMSF), pH 7.5. Cells were resuspended in NIM supplemented with Roche protease inhibitor mixture (1 tablet/20 ml). One ml of cell suspension was taken into a Dounce homogenizer and gently stroked for 8 -12 times; 0.2 ml of crude lysate was then transferred into the recording chamber coated with poly-L-lysine and isolated nuclei were left to attach at room temperature (ϩ24°C) for 5 min. This procedure was repeated every 40 -60 min for 5 h.

Patch clamp recordings
Single channel patch clamp recordings were obtained according to Mak et al. (53). We used equilibrium solutions with K ϩ as the charge carrier. For some initial experiments K ϩ was replaced with Cs ϩ , which is permeable for IP 3 channels but not for potassium channels (1 Data were filtered at 1000 Hz, digitized with Digidata 1550A (Axon Instruments) at 16-bit 2-kHz resolution and analyzed offline using PClamp 10 software. After formation of gigaseal (ϳ10 G⍀) and withdrawal of the pipette, excised inside-out configuration was established. This configuration allowed us to precisely control the solution composition at both luminal and cytoplasmic sides of the membrane. Each experiment was done at least four times with four nuclei in each different preparation. Only low-noise recordings with stable IP 3 Rs activity were taken into consideration. The conductance used for the majority (ϳ80%) of the recordings was 200 -220 pS. The success rate was 5% meaning that we detected active IP 3 responsive channels in 5% of patches with good gigaseal formation (no inhibitors added). When we used inhibitors, we observed 0% of active IP 3 responsive channels in Ͼ100 patches with good gigaseal formation. For the calculations of mean frequency, amplitude, open probability, and total power, we selected all events occurring in 60 s of representative recordings of samples in the presence or absence of phosphate compounds.

Cytosolic Ca 2؉ determinations
Fura 2 determinations were performed essentially as described before (8). After harvesting the cells, they were washed twice at 3000 ϫ g for 10 min at 4°C in buffer A, which contained (in mM): 116 NaCl, 5.4 KCI, 0.8 MgSO 4 , 5.5 D-glucose, and 50 Hepes, pH 7.2. Cells were resuspended in loading buffer consisting of buffer A plus 1.5% sucrose and 6 M Fura 2/AM. The suspension was incubated for 30 min in a 30°C water bath with mild agitation. The cells were then washed twice with ice-cold buffer A to remove extracellular dye. Cells were resuspended to a final density of 10 9 cells/ml in buffer A and were kept in ice. For fluorescence measurements, a 125 l sample of the cell suspension was diluted into 2.5 ml of buffer A (5 ϫ 10 7 cells/ml final density) in a cuvette placed in a thermostatically regulated (30°C) Hitachi F-7000 spectrofluorometer. Excitation was at 340 and 380 nm and emission was at 510 nm. The fura-2 fluorescence response to [Ca 2ϩ ] i concentration was calibrated from the ratio of 340/380 nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm as described by Grynkiewicz et al. (54). Other experimental conditions and calibrations were as described previously (8).

Statistical analysis
All values are expressed as mean Ϯ S.E., unless indicated. Differences between groups were compared using unpaired t-tests. Differences were considered statistically significant at p Ͻ 0.05, and n refers to the number of independent biological experiments. All statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software, San Diego, CA).