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Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada (L.-P.B., A.R.A., X.T., E.H., D.B., P.S.); and Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York University, New York, New York (Q.Z., D.E.L.)
Received for publication March 5, 2008.
Accepted for publication June 3, 2008.
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Abstract |
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). Among P2X receptors, the P2X1 subtype shows high sensitivity to the agonist 
,β-methylene ATP (,β-meATP); fast inactivation kinetics; and high expression in smooth muscle cells lining the urinary bladder, arteries, and vas deferens, where it regulates contractility (Sneddon, 2000; Burnstock, 2007). The activity of P2X1 receptor-channels can be modulated by phospholipase C-mediated signaling events. For example, recovery of P2X1 responses can be accelerated by activation of 1-adrenergic receptors in the vas deferens of guinea pig (Smith and Burnstock, 2004) or by activation of 5-HT2A (serotonin) receptors in the rat tail artery (Ase et al., 2005). In recombinant studies, P2X1 receptor currents have been shown to be potentiated by stimulation of coexpressed Gq-coupled metabotropic glutamate receptor1, P2Y
, and 5-HT2A receptors (Vial et al., 2004; Ase et al., 2005). By using cholesterol-depleting agents, Vial and Evans (2005) reported reduced ,β-meATP-induced contractions of the rat tail artery and decreased P2X1-mediated currents in transfected human embryonic kidney 293 cells, suggesting that lipids regulate P2X1 receptor signaling. Phosphoinositides have recently been shown to modulate P2X2 and P2X7 receptors (Fujiwara and Kubo, 2006; Zhao et al., 2007). Phosphoinositides are produced by specific lipid kinases through phosphorylation of phosphatidylinositol (PI) at the 3'-, 4'-, or 5'-position of the inositol ring. Phosphoinositides exert their regulatory role either indirectly as precursors of the phospholipase C-generated second messengers inositol 1,4,5-trisphosphate and diacylglycerol, or directly by interacting with proteins and thus modulating their localization, conformation, and activity. PI(4,5)P2, one of the most abundant phosphoinositides in the cell membrane, regulates many cellular events such as cytoskeleton remodeling, vesicle transport, and ion channel or transporter function (Suh and Hille, 2005; Di Paolo and De Camilli, 2006; Gamper and Shapiro, 2007).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Tong et al., 2005). In brief, rats were sacrificed by cervical dislocation, and the mesenteric arteries were isolated and collected in cold (4°C) Krebs' solution, pH 7.4, containing 118 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1 mM MgSO4, 1 mM KH2PO4, 25 mM NaHCO3, and 11 mM glucose. Arterial segments (approximately 2 mm in length) were transferred to a small superfusion chamber (5 ml; Living Systems Instrumentation, Inc., Burlington, VT), cannulated on a glass micropipette (
80 µm in diameter) at one end, sealed to another glass micropipette at the other end, and filled with oxygenated (5% CO2 and 95% O2) Krebs' solution (37°C; pH 7.4). Intraluminal pressure was maintained at 60 mm Hg, and vessels were superfused with Krebs' solution and allowed to stabilize and acquire basal tone. Online measurements of intraluminal diameter were performed using a closed circuit video system coupled with a video caliper. Direct abluminal application of 10 µM ,β-meATP was performed on vessels pretreated or not with the PI3/4-kinases inhibitor wortmannin (100 nM or 1 µM). In the wortmannin experiments, vessels were rapidly washed with fresh Krebs' solution before application of ,β-meATP. Expression of Recombinant P2X1 Receptors in Xenopus laevis Oocytes. Ovary lobes were surgically removed from X. laevis frogs deeply anesthetized by immersion in 0.2% Tricaine (Sigma-Aldrich, St. Louis, MO). After treatment with type I collagenase in calcium-free oocyte Ringer's medium for 2 h at room temperature, stage V to VI oocytes were manually defolliculated. Wild-type or mutant rat P2X1 subunit and rat 5-HT2A receptor cRNAs were transcribed in vitro using the mMessage mMachine kit (Ambion, Austin, TX) from pCDNA3 eukaryotic expression vector (Invitrogen, Carlsbad, CA) and then microinjected into the cytoplasm of oocytes. For expression of P2X1 alone, 25 ng of cRNA was injected in each oocyte. In coexpression experiments, 25 ng of P2X1 and 25 ng of 5-HT2A cRNA were injected. Oocytes were kept at 19°C in Barth solution containing 50 µg/ml gentamicin and 1.8 mM CaCl2 for 48 h before recording.
Electrophysiology. Two-electrode voltage-clamp recordings in X. laevis oocytes expressing P2X1 receptors were performed at room temperature using an OC-725C amplifier (Warner Instruments, Hamden, CT) and glass pipettes (1-3 M
) filled with 3 M KCl. Oocytes were placed in a 500-µl recording chamber and perfused at a flow rate of 10 to 12 ml/min with Ringer's solution, pH 7.4, containing 115 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES. Oocytes were held at -60 mV during recording. Unless specified, stimulations with 10 µM ATP (Sigma-Aldrich) dissolved in Ringer's solution were applied consecutively at 5-min intervals, and P2X1 responses were stable after the second application of ATP. When wortmannin was used, oocytes were incubated for 2 h in Barth's solution containing 100 nM or 35 µM wortmannin, or vehicle (0.3% dimethyl sulfoxide), before recording. For experiments involving coexpression of 5-HT2A receptors, 5-HT (1 µM) was applied in the bath for 5 min between the third and fourth ATP application. Potentiation index was defined as the ratio of the fourth over the third ATP-evoked P2X1 peak current. The phospholipids diC8-PI(4,5)P2 or diC8-PI(3,4,5)P3 (Echelon Biosciences Inc., Salt Lake City, UT) were injected (20 nl; 10 mM) in the cytoplasm of oocytes 30 min before final recording. For all the calculations of final drug concentration, we estimated the oocyte cell volume at 1 µl.
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were used. The internal (bath) solution contained 96 mM KCl, 0.5 mM EGTA, and 10 mM HEPES, pH 7.4. The external (pipette) solution contained also 1.8 mM CaCl2, as well as 20 µM ATP. Solutions were applied through a gravity-driven perfusion system. For each experiment, a minimum of two batches of oocytes were tested. Lipid Strip Binding Assay. Annealed oligonucleotides coding for a 16-amino acid sequence proximal to the C terminus of rat P2X1 (Ile356-Ala371) flanked by BamHI-EcoRI ends were subcloned into pGEX-2T vector for production of wild-type or mutant GST-P2X1 fusion proteins, before purification on glutathione-Sepharose resin. Lipid binding analysis of GST-P2X1 fusion proteins was conducted in vitro using identical amounts of phosphoinositides spotted on nitrocellulose membranes (PIP Strips; Echelon Biosciences Inc.), and the GST-N terminus of P2X1 or GST alone were used as negative controls. The membranes were blocked with TBS+T solution supplemented with 3% BSA for 1 h at room temperature, then incubated overnight with 5 µg/ml GST-fusion proteins in TBS+T with 3% BSA. The membranes were washed with TBS+T six times. Mouse anti-GST antibody (1:1000) was then added in TBS+T, 3% BSA solution for 1 h at room temperature. Repeated washes with TBS+T were followed by 1-h incubation with the secondary antibody horseradish peroxidase-labeled goat anti-mouse (1:5000) in TBS+T, 3% BSA at room temperature for detection of GST-P2X1 fusion protein binding with enhanced chemiluminescence.
Statistical Analysis. All the values are expressed as mean ± S.E.M. Data were analyzed using Student's t test or one-way analysis followed by Newman-Keuls multiple comparison tests, with p < 0.05 considered significant.
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Results |
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,β-meATP (10 µM) to vessel segments led to strong muscle constriction responses (69 ± 2% of maximal constriction induced by KCl, n = 6; Fig. 1). Micromolar concentrations of wortmannin, expected to inhibit both PI3-kinases and PI4-kinases, were used to test the impact of lower levels of phosphoinositides on native P2X1-mediated constrictive responses. When the mesenteric vessels were incubated with 1 µM wortmannin for 1 h, ,β-meATP-mediated constriction was significantly decreased to 28 ± 16% (n = 5) (Fig. 1B). However, no significant changes were measured at a lower (100 nM) concentration of wortmannin that inhibits PI3-kinases only (67 ± 9% of ,β-meATP-mediated constriction, n = 3; Fig. 1B).
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) and illustrated in Fig. 3A (see also Fig. 3D for quantitative results). However, 2 h of preincubation with 35 µM wortmannin blocked the accelerated recovery of P2X1 currents after 5-HT2A activation (Fig. 3, B and D). Lower concentration of wortmannin (100 nM) did not have any significant effect on 5-HT-induced modulation of P2X1 currents (Fig. 3, C and D). Rescue of P2X1 Current Responses and Kinetics by PI(4,5)P2. To study the effect of PI(4,5)P2 on P2X1 receptor-channels expressed in oocytes, we injected 200 µM diC8-PI(4,5)P2 in the cytoplasm 30 min before recording. Under basal conditions, ATP-evoked P2X1 current amplitudes and kinetics were not affected by the addition of diC8-PI(4,5)P2 (data not shown). However, in oocytes preincubated with 35 µM wortmannin, diC8-PI(4,5)P2 addition led to a significant rescue of P2X1 current amplitudes: 2.8 ± 0.5 µA(n = 8) compared with 1.0 ± 0.2 µA in PBS-injected oocytes (n = 14) (Fig. 4, A and B). Thus, whereas addition of diC8-PI(4,5)P2 did not seem to increase ATP-evoked P2X1 currents under basal conditions, it did so when endogenous levels of PI(4,5)P2 were previously depleted by wortmannin treatment. In contrast, 200 µM diC8-PI(3,4,5)P3 injections did not have any rescuing effect on the current amplitudes of P2X1 receptors after wortmannin treatment (Fig. 4B).
In addition, we found that PI(4,5)P2 levels have a significant impact on the kinetics of P2X1 current responses. Thus, as illustrated by the current traces in Fig. 5A, treatment with high concentration of wortmannin clearly slowed down both the 10 to 90% rise time of the activation phase and the inactivation time (one-exponential fit) of P2X1 current responses. The 10 to 90% rise time was 0.17 ± 0.01 s (n = 9) in control conditions and 0.25 ± 0.03 s (n = 9) after wortmannin. The time constant (
) of inactivation was 0.92 ± 0.07 s (n = 9) in control conditions and 1.42 ± 0.14 s (n = 9) after wortmannin. We observed that injection of PI(4,5)P2 completely restored the kinetic parameters of P2X1 responses to their control levels (Fig. 5, B and C).
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2-fold) for P2X1 currents in the presence of diC8-PI(4,5)P2, compared with rundown baseline levels (Fig. 6B).
The Single Lysine Lys364 in the C Terminus of P2X1 Subunit Is a Critical Determinant for Binding Phosphoinositides. Because phosphoinositides are anionic, we tested for potential interactions of these lipids with positively charged residues on the intracellular domains of the P2X1 subunits by performing a lipid strip assay. The proximal region of the C-terminal domain displays a cluster of basic amino acids (Fig. 7A) that are potentially involved in the interaction with phosphoinositides, as recently reported for the P2X2 receptor (Fujiwara and Kubo, 2006; Zhao et al., 2007). No specific binding was observed when a GST-P2X1 fusion protein expressing the N-terminal peptide Phe12-Val29 was tested (Fig. 7B). However the GST-P2X1 fusion protein containing the C-terminal peptide Ile356-Ala371 selectively bound to several phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3, phosphatidylserine, and to a lesser extent, PI(3,4)P2 and phosphatidic acid, as illustrated in Fig. 7B. We investigated the contribution of each lysine or arginine residue in the C-terminal peptide by replacing them with the neutral residue glutamine. Only one substitution, K364Q, led to the complete suppression of binding (Fig. 7B); therefore, the lysine Lys364 is a critical determinant for interaction with phosphoinositides. As shown in Fig. 7C, ATP-evoked current amplitude was smaller in oocytes expressing the mutant receptor P2X1 K364Q (4.94 ± 0.69 µA; n = 10) compared with wild-type receptor (8.27 ± 0.99 µA; n = 10). Moreover, P2X1 K364Q receptors showed higher sensitivity to depletion of PI(4,5)P2 induced by 35 µM wortmannin (13.70 ± 1.80% of control response; n = 19) than wild-type receptors (44.88 ± 10.13% of control response; n = 9), confirming a decrease in PI(4,5)P2 binding affinity (Fig. 7D).
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Discussion |
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; Hansen et al., 1998). This purinergic component of smooth muscle contraction is present in rodents (Mulryan et al., 2000) as well as in humans (Banks et al., 2006). In the present study, we demonstrated the modulation of P2X1 responses by phosphoinositides and its physiological relevance because ,β-meATP-evoked P2X1-mediated constrictions of the rat mesenteric artery are sensitive to the pharmacological depletion of PI(4,5)P2. We were able to reconstitute in the X. laevis oocyte expression system the modulation of P2X1-mediated responses by blocking the critical lipid kinases involved in phosphoinositides synthesis, thus indicating that ubiquitous intracellular mechanisms are involved. Two-electrode voltage-clamp recordings showed decreased ATP-evoked P2X1 current responses with high (micromolar) concentration of wortmannin, required to block both PI4-kinases and PI3-kinases (Nakanishi et al., 1995), whereas low (nM) concentrations of wortmannin, known to block PI3-kinases only, did not have any effect. The inhibitory effect of wortmannin is not due to a decrease in sensitivity of P2X1 receptors to ATP because 1) the inhibition was unsurmountable at supramaximal concentrations of ATP, 2) maximal current amplitude was reached at 10 µM ATP after wortmannin treatment as in control conditions, and 3) wortmannin had no effect on P2X1 currents induced by 0.1 µM ATP. We can conclude that ATP becomes a partial agonist for P2X1 when PI(4,5)P2 levels are depleted. In other words, minimal PI(4,5)P2 levels are required for the expression of a full P2X1 response to ATP. Recovery of P2X1 responses was also found sensitive to treatment with high concentration of wortmannin, suggesting that desensitization of P2X1 receptor-channels is regulated by PI(4,5)P2.
We and others have reported the potentiation of P2X1 responses by activation of Gq-coupled receptors (Vial et al., 2004; Ase et al., 2005), so we tested the impact of wortmannin on the modulation of P2X1 responses by Gq-coupled 5-HT2A receptors. The lack of potentiation of P2X1 responses after blockade of PI4-kinases by 35 µM wortmannin, as well as the absence of effect of PI3-kinases blockade by 100 nM wortmannin, supported a critical role for PI(4,5)P2 as phospholipase C substrate in the cross-talk between 5-HT2A and P2X1 receptors. The effectiveness of the blockade of PI4-kinases and PI(4,5)P2 synthesis was confirmed by the suppression of inositol 1,4,5-trisphosphate-induced Ca2+-dependent chloride currents, triggered by 5-HT, after treatment with 35 µM wortmannin. Stimulation of the phospholipase C-coupled platelet-derived growth-factor tyrosine kinase receptor has been shown to decrease PI(4,5)P2-sensitive P2X7 currents (Zhao et al., 2007); however, we did not observe any decrease of P2X1 responses after 5-HT2A stimulation. This suggests that a transient stimulation of Gq-coupled receptors does not significantly deplete the steady-state levels of PI(4,5)P2 available at the plasma membrane, in agreement with what is known about the high turnover rate of this phospholipid (Hilgemann, 2007).
The specific involvement of PI(4,5)P2 in modulating P2X1 channels responses is directly supported by the fact that addition of PI(4,5)P2, and not PI(3,4,5)P3, significantly rescued the amplitude of P2X1 currents after blockade of PI4-kinases with 35 µM wortmannin. Decreased levels of phosphoinositides not only affected the amplitude of P2X1 currents but also their kinetics, because wortmannin clearly slowed down their 10 to 90% rise time and increased their time constant of inactivation. Addition of intracellular PI(4,5)P2 after treatment with wortmannin normalized the kinetics of the P2X1 currents back to their control values. Under basal conditions, however, increasing PI(4,5)P2 levels by direct injection into the cytoplasm affected neither basal P2X1 current amplitudes nor activation/inactivation kinetics. Thus, at least in oocytes, endogenous PI(4,5)P2 levels saturate the P2X1 receptor-channels expressed at the cell surface.
Our results using inside-out macropatches excised from P2X1-expressing oocytes provided evidence for the sensitivity of P2X1 receptor-channels to direct application of PI(4,5)P2. P2X1 shows little activity upon patch excision because of a rapid rundown resulting from receptor desensitization and dephosphorylation of PI(4,5)P2 by lipid phosphatases (Huang et al., 1998; Zhang et al., 1999). Direct regulation of channel activity by phosphoinositides in excised patches has been reported for several types of ion channels, including the prototypical Kir and KCNQ channels also modulated by PI(4,5)P2 (Lopes et al., 2002; Suh and Hille, 2002, 2005; Zhang et al., 2003). Application of exogenous PI(4,5)P2 on the intracellular side of the patch rescued P2X1 currents from rundown, indicating the requirement of PI(4,5)P2 for full P2X1 function and arguing against trafficking to the surface as the mechanism for PI(4,5)P2-induced potentiation of P2X1 currents.
Searching for phospholipid-binding motifs in the intracellular sequences of P2X1 subunits, we confirmed the direct binding of PI(4,5)P2 to the C-terminal domain Ile356-Ala371. This domain proximal to the second transmembrane domain, in a position favorable for interactions with the anionic head groups of phospholipids according to the transmembrane topology of P2X subunits, contains a high density of basic residues (five lysines and one arginine), and we have found that the single lysine at position 364 plays a critical role in these interactions. The mutant receptor P2X1 K364Q remains functional but with a lower binding affinity for PI(4,5)P2, indicated by a higher sensitivity to wortmannin-induced depletion, analogously to mutants of other PI(4,5)P2-sensitive channels (Rohács et al., 2005). The fact that the K364Q mutation in the functional P2X1 receptor-channel did not lead to complete loss of PI(4,5)P2 binding and insensitivity to wortmannin suggests that other determinants contribute to phosphoinositides binding. It is noteworthy that the C-terminal sequence of P2X1 also showed strong affinity for other anionic phospholipids. PI(4,5)P2 and PI(4)P are by far the most abundant phosphoinositides in the plasma membrane; however, the dependence of ion channel activity on other phosphoinositides is well documented (Zhainazarov et al., 2004; Pochynyuk et al., 2005; Srivastava et al., 2005). A similar but qualitatively distinct lipid-binding profile was recently reported for the homologous C-terminal sequence of P2X2 (Fujiwara and Kubo, 2006). Therefore, this short domain seems to be of general importance for the regulation of P2X function by phosphoinositides.
In conclusion, we have provided strong evidence that the phospholipid PI(4,5)P2 is a physiological ligand of P2X1 receptor-channels, and our results suggest a novel mode of regulation of ionotropic P2X1 responses by the various metabotropic pathways linked to phosphoinositide metabolism.
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Footnotes |
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L.-P.B. and A.R.A. contributed equally to this work.
ABBREVIATIONS: ,β-meATP, ,β-methylene ATP; 5-HT, 5-hydroxytryptamine; PI, phosphatidylinositol; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; diC8, 1,2-dioctanoyl-sn-glycerol; GST, glutathione transferase; TBS+T, Tris-buffered saline/Tween 20; BSA, bovine serum albumin; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.
Address correspondence to: Dr. Philippe Séguéla, Montreal Neurological Institute, 3801 University, Suite 778, Montreal, QC, Canada H3A 2B4. E-mail: philippe.seguela{at}mcgill.ca
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