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The Journal of Neuroscience, November 26, 2008, 28(48):12938-12945; doi:10.1523/JNEUROSCI.3038-08.2008

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Cellular/Molecular
Phosphoinositides Regulate P2X₄ ATP-Gated Channels through Direct Interactions

Louis-Philippe Bernier,¹ Ariel R. Ase,¹ Stéphanie Chevallier,¹ Dominique Blais,¹ Qi Zhao,² Éric Boué-Grabot,³ Diomedes Logothetis,² and Philippe Séguéla¹

¹Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montréal, Québec, Canada H3A 2B4, ²Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York University, New York, New York 10029, and ³Université de Bordeaux 2, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5227, 33076 Bordeaux, France

	Abstract

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P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X₄ subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X₄ function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P₂ (PIP₂) and PI(3,4,5)P₃ (PIP₃), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X₄ receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP₂ and PIP₃ with wortmannin significantly decreased P2X₄ current amplitude and P2X₄-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X₄ responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP₂ on excised inside-out macropatches rescued P2X₄ currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X₄ subunit (Cys₃₆₀–Val₃₇₅) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP₂ and PIP₃ on P2X₄ function through direct channel–lipid interactions.

Key words: P2X; purinergic; PIP₂; calcium; microglia; neuropathic pain

	Introduction

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Mammalian P2X receptors are ATP-gated nonselective cation channels, generated from the trimeric assembly of homologous subunits (P2X_1–7). Each subunit is composed of two transmembrane domains connected by a large ectodomain, with both N and C termini facing the intracellular side of the plasma membrane (North, 2002). In the nervous system, P2X receptor channels are expressed at the plasma membrane of neurons, astrocytes, and microglia, where they contribute to intercellular purinergic communication.

The P2X₄ subunit forms a functional homomeric channel with high permeability to calcium ions (Egan and Khakh, 2004). It can also heteromerize with P2X₁ (Nicke et al., 2005), P2X₆ (Lê et al., 1998), or P2X₇ subunits (Guo et al., 2007). It is expressed at high levels in central neurons and in microglial cells (Lê et al., 1998; Rubio and Soto, 2001; Tsuda et al., 2003). In recent years, microglial P2X₄ receptor channels have been found to play a critical role in the development of neuropathic pain induced by peripheral nerve injury (Tsuda et al., 2003; Inoue et al., 2004; Trang et al., 2006). The upregulation of P2X₄ expression in activated microglia following nerve injury is critical to the development of tactile allodynia (Tsuda et al., 2003) via the release of microglial BDNF and KCC2 cotransporter-mediated disinhibition of dorsal horn neurons (Coull et al., 2003, 2005). Despite the involvement of the P2X₄ receptor subtype in the pathogenesis of chronic pain, very little is known about its posttranslational regulation.

A lot of attention has been brought to phosphoinositides as modulators of membrane proteins. These phospholipids are produced by the combinatorial action of selective lipid kinases, namely phosphoinositol 3-kinase (PI3K), phosphoinositol 4-kinase (PI4K) and phosphoinositol 5-kinase, which phosphorylate the inositol ring at the 3', 4', or 5' positions. Synthesis of the major phosphoinositide PI(4,5)P₂ (PIP₂) is completed through successive phosphorylations by PI4K and phosphoinositol(4)5-kinase. Formation of PI(3,4,5)P₃ (PIP₃) from PIP₂ necessitates one additional phosphorylation by PI3K. Phosphoinositides are regulators of several important cellular processes, such as vesicle trafficking, cytoskeleton remodeling, and ion channel function (Hilgemann et al., 2001; Suh and Hille, 2005). Interestingly, it has been shown that P2X₁, P2X₂, and P2X₇ receptors are positively modulated by different phosphoinositides (Fujiwara and Kubo, 2006; Zhao et al., 2007; Bernier et al., 2008).

Phosphoinositides can interact with membrane proteins via electrostatic binding to positively charged residues (Rosenhouse-Dantsker and Logothetis, 2007). The P2X₄ subunit contains a phosphoinositide-binding motif characterized by a cluster of lysine and arginine residues in the intracellular C-terminal domain in close proximity to its second transmembrane domain. In this study, we used electrophysiological techniques to elucidate the role major phosphoinositides play in the modulation of microglial and recombinant P2X₄ receptor channel function. We also investigated the impact of phosphoinositides on P2X₄-mediated calcium entry and the structural characteristics of their interactions with P2X₄ using a lipid binding assay.

	Materials and Methods

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Patch-clamp recordings in BV-2 cells. Mouse BV-2 microglial cells were a kind gift from Dr. Jean Labrecque (AstraZeneca R&D) and were routinely maintained at 37°C and 5% CO₂ in DMEM supplemented with 5% heat-inactivated FBS, 10 mM pyruvate (Sigma), 5% L-glutamine (Invitrogen), penicillin, and streptomycin. Whole-cell patch-clamp recordings of BV-2 cells (V_hold = –60 mV) were performed using pipettes filled with internal solution, pH 7.2, containing (in mM) 120 K-gluconate, 1 MgCl₂, 4 NaOH, and 10 HEPES. The normal recording solution, pH 7.4, comprised (in mM) 130 NaCl, 5 NaOH, 3 KCl, 1 MgCl₂, 2 CaCl₂, and 10 HEPES. Low divalent recording solution contained 0.3 mM CaCl₂ without Mg²⁺. Membrane currents were recorded using an Axopatch 200B amplifier and digitized at 500 Hz with a Digidata 1330 interface (Axon Instruments). Drugs were dissolved in recording solution and applied using a SF-77B fast perfusion system (Warner Instruments) at a rate of 1 ml/min. All experiments were performed at room temperature.

Ratiometric measurement of [Ca²⁺]_i in BV-2 cells. BV-2 cells were loaded with Ca²⁺-sensitive fluorescent dye fura-2 AM (5 µM, Molecular Probes) in Ringer's solution (in mM: 130 NaCl, 3 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES; 37°C, pH 7.4) with 1% BSA for 30 min at 37°C in the dark. Cells were washed three times to remove extracellular fura-2 AM and left for at least 30 min for complete hydrolysation of acetoxymethyl ester before imaging. Fluorescence measurement of Ca²⁺ in individual cells were performed on an inverted TE2000-U microscope (Nikon) equipped with 40x oil-immersion objective [CFI super(S) fluor, Nikon]. Fura-2 AM was excited alternatively at 340 and 380 nm with a Lambda 10-2 filter wheel (Sutter Instrument) coupled to a xenon arc lamp (XPS-100, Nikon). The fluorescent emission was monitored at 510 nm and recorded by a high-resolution cooled CCD camera (Cool Snap-HQ, Roper Scientific/Photometrics) interfaced to a Pentium III PC. Pairs of 340 and 380 nm images were acquired every second and ratio images were calculated using Metafluor 7.0 software (Molecular Devices). The experiment was performed at room temperature. Cells were superfused with Ringer's solution and drugs at a flow rate of 1 ml/min. Results were expressed as the 340/380 nm emission ratio proportional to the intracellular calcium. The magnitude of responses to ATP was calculated as the difference between the peak of the response and the basal ratio ( ratio). To test the viability of the cells, they were challenged with 1 µM ionomycin at the end of each recording. In each experiment, all conditions were tested (control, ivermectin, and ivermectin plus wortmannin) and data were collected from 3 independent experiments.

Two-electrode voltage clamp recordings in Xenopus oocytes. Oocytes were surgically removed from Tricaine-anesthetized female Xenopus laevis frogs and were incubated in OR2 solution containing 1–2 mg/ml type IA collagenase at room temperature for 2 h under vigorous agitation. Stage V and VI oocytes were then manually defolliculated before intranuclear microinjection of plasmid DNA coding for rP2X₄ (3 ng). After injection, oocytes were incubated in Barth's solution containing 1.8 mM CaCl₂ at 19°C for 24 to 72 h before electrophysiological recordings. Two-electrode voltage-clamp recordings (V_hold = –60 mV) were performed using glass pipettes (1–3 M) filled with 3 M KCl solution. Oocytes were placed in a recording chamber and perfused at a flow rate of 10 to 12 ml/min with Ringer's solution, pH 7.4, containing (in mM) 115 NaCl, 5 NaOH, 2.5 KCl, 1.8 CaCl₂, and 10 HEPES. Membrane currents (d.c., 1 kHz) were recorded using a Warner OC-725B amplifier (Warner Instrument) and digitized at 500 Hz. Agonists were dissolved in the perfusion solution and applied using a computer-driven valve system. All series of recordings consisted of three successive applications of ATP 100 µM, with a 4 min washout with Ringer's solution between each application.

Macropatch recordings in Xenopus oocytes. Inside-out macropatch experiments were performed using an Axopatch 200B amplifier (Axon Instruments). PClamp 9 was used for data acquisition and analysis. The vitelline membrane of Xenopus oocytes was removed using fine forceps before recordings. Electrodes with resistance of 0.5–1.0 M were used. The currents were evoked by ramps from +100 mV to –100 mV. The time course of the currents at –80 mV was analyzed. The pipette solutions contained 100 µM ATP and dioctanoyl (diC₈)-PIP₂ (5 µM) was dissolved in the bath solution. The solutions were applied through a gravity-driven perfusion system. For each experiment, a minimum of two batches of oocytes were tested.

Lipid binding assay. Oligonucleotides coding for amino acid sequences proximal to the C terminus (C₃₆₀–V₃₇₅) or the N terminus (F₁₂–V₂₉) of P2X₄ were inserted into the pGEX-2T vector (New England Biolabs) for the production of the corresponding recombinant GST fusion proteins. The lipid binding analysis of the GST-fusion proteins was conducted using lipid coated hydrophobic membranes (PIP Strips, Echelon Biosciences). The membranes were first blocked with TBS+T solution supplemented with 3% BSA for 1 h at room temperature. The membranes were then incubated overnight in TBS+T with 3% BSA and 1 µg/ml GST fusion protein. The membranes were then washed with TBS+T six times, 5 min per wash. The primary antibody (mouse anti-GST, 1:1000) was then added in TBS+T, 3% BSA solution for 1 h. Washes with TBS+T were repeated, followed by incubation with the secondary antibody (goat anti-mouse HRP, 1:5000) in TBS+T, 3% BSA. The membranes were washed again in TBS+T, then bound proteins were detected in ECL.

Data analysis. Peak currents, defined as the maximal amplitude recorded during agonist application, were measured for comparative analysis. For recovery experiments, the amplitude of the third response was compared with the first and expressed as a percentage. The results obtained after a 2 h drug incubation were always compared with those obtained after a 2 h control incubation in Barth's solution containing vehicle (100 nM or 35 µM DMSO). For current kinetics experiments, activation rate was measured as the rise time (in seconds) from 10% to 90% of the peak amplitude. For desensitization rate, the time constant was measured with a one-exponential fit of the inactivation phase of the current. Data are presented as mean ± SEM and analyzed using Student's t test or one-way ANOVA followed by a Bonferroni's multiple comparison test. Normalized data were analyzed using nonparametric Mann–Whitney test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Depletion of phosphoinositides decreases P2X₄ current density in BV-2 microglial cells
To assess the role phosphoinositides might play on native P2X₄ currents, we tested the effect of blocking PIP₂ and/or PIP₃ synthesis with wortmannin in BV-2 microglial cells. The furanosteroid wortmannin has been extensively used to block key lipid kinases in the phosphoinositides synthesis pathways. It is a potent inhibitor of PI3K at nanomolar concentrations while it blocks also PI4K at micromolar concentrations (Nakanishi et al., 1995; Vanhaesebroeck et al., 2001). Murine BV-2 cells express endogenous P2X₄ and P2X₇ channels (Raouf et al., 2007). To isolate P2X₄ currents from P2X₇ currents, we took advantage of the differential agonist sensitivity of the mouse P2X₄ and P2X₇ subtypes. Mouse P2X₇ receptors exhibit a markedly lower sensitivity to the agonist ATP than the mouse P2X₄ subtype (ATP EC₅₀ >500 µM and <10 µM respectively) (Townsend-Nicholson et al., 1999). Ivermectin is an allosteric modulator of P2X₄ receptors that enhances currents by stabilizing the agonist-induced open state (Priel and Silberberg, 2004). Therefore, ivermectin provides an additional tool to isolate P2X₄ currents from microglial cells. Application of 100 µM ATP evoked ivermectin-potentiated inward currents typical of the P2X₄ subtype (Fig. 1A). Vehicle-treated cells showed an ATP-evoked current density of 1.7 ± 0.3 pA/pF, while current density following 3 µM ivermectin treatment was increased to 13.9 ± 2.2 pA/pF (p < 0.0001, n = 9–10) (Fig. 1A,B). Depletion of PIP₃ levels with 100 nM wortmannin (PI3K blockade) for 2 h led to a significant decrease of ivermectin-potentiated P2X₄ current density (6.3 ± 0.6 pA/pF; p = 0.0096, n = 7). A comparable decrease was observed by blocking both PI3K and PI4K with 35 µM wortmannin for 2 h when compared with ivermectin alone (4.2 ± 0.9 pA/pF; p = 0.0025, n = 7).

View larger version (20K): [in this window] [in a new window]   Figure 1. Blocking phosphoinositide synthesis with wortmannin leads to a decrease in P2X4 current amplitude in microglial BV-2 cells. BV-2 cells were stimulated with 100 µM ATP to evoke inward P2X4 currents. A, Sample traces of P2X4 currents after BV-2 cells were treated with vehicle (DMSO), 3 µM ivermectin, or 3 µM ivermectin with 100 nM or 35 µM wortmannin. B, Ivermectin treatment led to an increase in current density, the means before and after ivermectin were 1.70 ± 0.27 and 13.90 ± 2.19 pA/pF, respectively (p < 0.0001, n = 9–10). Treatment with 100 nM or 35 µM wortmannin led to a decrease in current density to 6.25 ± 0.58 pA/pF (p = 0.0096, n = 7) and 4.21 ± 0.94 pA/pF (p = 0.0025 n = 7), respectively.

 
P2X₄-mediated calcium entry is sensitive to depletion of phosphoinositides
Taking advantage of the fact that P2X₄ receptor channels are highly calcium permeable (Egan and Khakh, 2004), we used ratiometric fura-2 fluorescence imaging to measure changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in BV-2 cells. Application of 100 µM ATP for 30 s induced a calcium response that was evaluated as the difference between the fluorescence ratio at the peak of the response and the basal ratio ( ratio) (Fig. 2A). In 77 cells recorded in 3 independent experiments, the increase of [Ca²⁺]_i corresponded to a mean ratio of 0.24 ± 0.02 (Fig. 2A,D). Applications of 1 µM ionomycin evoked typical and reproducible rises of [Ca²⁺]_i. Given that ivermectin selectively enhanced ATP-evoked P2X₄-current densities, we then examined the sensitivity of ATP-induced Ca²⁺ ion entry to ivermectin. Two minutes pretreatment with 3 µM ivermectin strongly potentiated the calcium response to 100 µM ATP ( ratio = 0.55 ± 0.05, n = 71) (Fig. 2B,D). The rise of fluorescence ratio induced by ATP was increased by 229%. Depletion of PIP₂ and PIP₃ levels by preincubation with 35 µM wortmannin (PI3K and PI4K blockade) for 10 min significantly decreased the P2X₄-mediated calcium entry ( ratio: 0.32 ± 0.04, n = 45) (Fig. 2C,D). The responses to ionomycin were similar in BV-2 cells exposed to wortmannin plus ivermectin compared with those exposed to ivermectin alone ( ratio = 0.24 ± 0.02, n = 45 and 0.28 ± 0.02, n = 71, respectively). Using published calibration methods (Grynkiewicz et al., 1985), we estimated the rise of P2X₄-mediated [Ca²⁺]_i at 240 ± 10 nM for vehicle-treated cells, 650 ± 70 nM for cells treated with 3 µM ivermectin, and 360 ± 30 nM for cells treated with both 3 µM ivermectin and 35 µm wortmannin. Altogether, these results show that disrupting PIP₂ and PIP₃ levels significantly decrease the capacity of P2X₄ channels to mediate calcium entry into microglial cells.

View larger version (27K): [in this window] [in a new window]   Figure 2. Wortmannin treatment leads to a decrease in ATP-induced calcium entry in BV-2 cells. A–C, Representative recordings of calcium-mediated fura-2 fluorescence (340/380 nm ratio) over time in BV-2 cells in response to 100 µM ATP. A, In control conditions, a BV-2 cell was challenged with 100 µM ATP during 30 s. B, Before ATP application, 2 min preapplication of 3 µM ivermectin strongly increased the ATP-induced [Ca2+]i increase. C, Ten minutes of preincubation of BV-2 cell with 35 µM wortmannin led to a decrease in ATP-induced calcium entry. Cells were challenged with 1 µM ionomycin to test their viability at the end of recordings. ATP, ivermectin, and ionomycin were applied by superfusion at the times indicated by the horizontal bars. D, Quantitation of the amplitude of responses to 100 µM ATP, measured by the ratio (see Materials and Methods). Error bars represent mean ± SEM of 3 independent experiments. White bar: mean amplitude of ATP response under control conditions (n = 77). Black bar: mean amplitude of ATP response after perfusion with 3 µM ivermectin (n = 71). Gray bar: mean amplitude of ATP response after preincubation with 35 µM wortmannin (gray bar, n = 45) and perfusion with 3 µM ivermectin. Significance was determined by ANOVA using Bonferroni's multiple comparison test. ***p < 0.001.

 
P2X₄ current amplitude is decreased by wortmannin treatment in Xenopus oocytes
To further investigate the modulation of P2X₄ currents by phosphoinositides, we conducted experiments on Xenopus oocytes expressing recombinant P2X₄ receptor channels. Oocytes were incubated for 2 h in wortmannin at 100 nM (PI3K blockade), or at 35 µM (PI3K and PI4K blockade). The amplitude of the first evoked P2X₄ current was recorded (Fig. 3). The average current amplitude in control conditions was 4.2 ± 0.6 µA (n = 6). As shown in Figure 3, A and B, 100 nM wortmannin induced a significant decrease of 50% in the P2X₄ current amplitude, down to 2.1 ± 0.6 µA (p = 0.0347, n = 6), whereas 35 µM wortmannin led to an even greater inhibition of P2X₄ current amplitude (0.8 ± 0.4 µA; p = 0.0007, n = 7). Dose–response curves confirmed a fourfold decrease in maximal current amplitudes after treatment with wortmannin (Fig. 3C). They also revealed a small albeit significant increase in the EC₅₀ of ATP (18.7 ± 1.8 µM in vehicle-treated oocytes, 28.7 ± 2.4 µM in wortmannin-treated oocytes, n = 4–9, *p < 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 3. Blocking phosphoinositides synthesis leads to a decrease in P2X4 current amplitude in Xenopus oocytes. A, Sample traces showing ATP-gated currents in oocytes expressing P2X4 after vehicle or 100 nM or 35 µM wortmannin treatment. B, Quantitative results. P2X4 currents have an average amplitude of 4.17 ± 0.64µA (n = 6) in oocytes incubated in control solution (Barth's solution with DMSO) while blocking PI3K with 100 nM wortmannin decreased P2X4 current amplitude to 2.09 ± 0.57 µA (p = 0.0347, n = 6). Blocking both PI3K and PI4K with 35 µM wortmannin decreased current amplitudes to 0.83 ± 0.38 µA (p = 0.0007, n = 7). C, Normalized ATP concentration-current amplitude curves in absence (vehicle) or in presence of 35 µM wortmannin. Wortmannin decreases the maximal current amplitude and increases the EC50 of ATP (n = 4–9).

 
Impact of phosphoinositides on the speed of recovery of P2X₄ currents
We then tested whether the depletion of PIP₂ or PIP₃ could affect the speed of recovery of P2X₄-mediated currents. We activated the receptor with three successive applications of 100 µM ATP at 4 min intervals, and measured the amplitude of the third current relative to the first current response, defined as the third/first recovery ratio. Under control conditions, a 4 min washout between each agonist application was enough for the receptor to fully recover: the third/first ratio was of 100 ± 8% (n = 7) (Fig. 4A). Blocking PIP₃ synthesis with 100 nM wortmannin preincubation led to a gradual decrease of the currents, the amplitude of the third currents being 66 ± 11% of that of the initial current (Fig. 4B). When both PIP₂ and PIP₃ levels were depleted with 35 µM wortmannin preincubation, the third current went down to 26 ± 5% of the first one (Fig. 4C). The third/first ratios under both 100 nM and 35 µM wortmannin were significantly smaller than under control conditions (p = 0.0251, n = 6 and p < 0.0001, n = 7, respectively).

View larger version (16K): [in this window] [in a new window]   Figure 4. Phosphoinositides depletion leads to decreased recovery of P2X4 currents during successive activations with ATP. In Xenopus oocytes expressing P2X4, we measured the currents induced by three successive applications of 100 µM ATP, at 4 min intervals. Rundown of currents is measured as the ratio of the amplitude of the third response over that of the first. Shown here are sample traces of a recording series after incubation in vehicle (A), 100 nM wortmannin (B), and 35 µM wortmannin (C). D, Under control conditions, the third current amplitude was 100.4 ± 8.3% (n = 7) of the first, i.e., complete recovery of the receptor was observed. After 2 h incubation in 100 nM wortmannin, currents recovered to 65.8 ± 10.8% (p = 0.025, n = 6) and to 26.3 ± 4.7% (p < 0.001, n = 7) after 35 µM wortmannin incubation.

 
Injection of phosphoinositides rescues P2X₄ current amplitude in wortmannin-treated oocytes
To test the selective involvement of phosphoinositides in the modulation of P2X₄ channel responses by wortmannin, we conducted experiments in which diC₈-PIP₂ or diC₈-PIP₃ were injected into the oocyte cytoplasm. Initial channel activation was recorded, followed by injection of vehicle (PBS) or phosphoinositides, to reach an intracellular concentration of 200 µM. The subsequent current activation was then recorded and compared with preinjection amplitude. Notably, under basal conditions, injection of either diC₈-PIP₂ or diC₈-PIP₃ did not lead to any change in the P2X₄ current amplitude (data not shown), suggesting that PIP₂ and PIP₃ levels are saturating under basal conditions. However, injection of phosphoinositides rescued P2X₄ current amplitudes in conditions of wortmannin-induced depletion (Fig. 5A,B). As shown in Figure 5C, diC₈-PIP₃ injection induced a 396 ± 64% increase in P2X₄ (p = 0.012, n = 4), whereas diC₈-PIP₂ injection led to a 827 ± 244% increase (p = 0.006, n = 4).

View larger version (16K): [in this window] [in a new window]   Figure 5. Injection of PIP2 and PIP3 rescues P2X4 current amplitude under conditions of wortmannin-induced phosphoinositide depletion. Oocytes expressing P2X4 were incubated in 35 µM wortmannin, and ATP-evoked currents were then recorded before and after phosphoinositide injection. Sample traces showing the effect of the injection of diC8-PIP3 (A) or diC8-PIP2 (B) compared with the preinjection current. C, Quantitative data show that, after phosphoinositide depletion with wortmannin, injection of diC8-PIP3 leads to a 396 ± 64% (p = 0.012, n = 4) increase of the P2X4 response to 100 µM ATP, and injection of diC8-PIP2 increases the currents by 827 ± 244% (p = 0.006, n = 4). Injection of vehicle (PBS) did not induce any significant change on the P2X4 currents (n = 7).

 
P2X₄ current kinetics is sensitive to PIP₂ levels
To assess changes in P2X₄ current kinetics in Xenopus oocytes, we examined the activation rate, calculated as the 10–90% rise time of the onset current, and the desensitization rate calculated as the mono-exponential fit of the inactivation current phase (Fig. 6A). Preincubation with 35 µM wortmannin (PI3K and PI4K blockade) induced an increase of the 10–90% rise time (1.15 ± 0.1 s, p = 0.0068, n = 7) compared with vehicle-treated oocytes (0.72 ± 0.08 s, n = 10) indicative of a slower activation rate of P2X₄ channels (Fig. 6B,C). The 10–90% rise time was not affected by treatment with 100 nM wortmannin (PI3K blockade) (0.59 ± 0.06 s, n = 7). The same kinetics parameter was studied at 35 µM wortmannin followed by injection of diC₈-PIP₂ or vehicle. Under these conditions, cytoplasmic diC₈-PIP₂ injection led to a faster activation rate compared with vehicle-injected oocytes, with respective rise times of 0.90 ± 0.07 s and 1.61 ± 0.28 s respectively (p = 0.0263, n = 13–14).

View larger version (22K): [in this window] [in a new window]   Figure 6. Phosphoinositide depletion leads to changes in the kinetics of P2X4 currents in Xenopus oocytes. A, Sample traces showing the typical kinetics of P2X4 currents, under control conditions and after a 2 h treatment with 35 µM wortmannin. B, Quantitative results for P2X4 current 10–90% rise times. Incubation of the oocytes in 35 µM wortmannin, but not in 100 nM wortmannin, led to an increase in the rise time (p = 0.0069, n = 7–10) and injection of diC8-PIP2 normalized the rise time (p = 0.026, n = 13–14). C, Quantitative results for the desensitization time constants. Incubation in 35 µM wortmannin, but not in 100 nM wortmannin, led to an increase in the desensitization time (p = 0.0089, n = 5–6). After incubation in 35 µM wortmannin, injection of diC8-PIP2 decreased the desensitization time significantly compared with the injection of vehicle (PBS) (p < 0.0001, n = 13–17).

 
The desensitization rate of P2X₄-mediated currents was studied under similar experimental conditions. Vehicle-treated oocytes showed a desensitization rate of 6.73 ± 0.85 s, whereas preincubation with 35 µM wortmannin slowed it significantly to 15.76 ± 2.8 s (p = 0.0089, n = 5–6). Under these conditions, cytoplasmic diC₈-PIP₂ injection led to a faster desensitization rate compared with that of vehicle-injected oocytes, with inactivation times of 5.15 ± 0.34 s and 9.17 ± 0.59 s respectively (p < 0.0001, n = 13–17). A treatment with 100 nM wortmannin did not modify the desensitization rate of P2X₄ currents (6.23 ± 1.96 s, n = 6).

PIP₂ activates P2X₄ current responses in excised inside-out macropatches
Excised inside-out patch recordings were performed to test the direct effects of phosphoinositides on heterologously expressed P2X₄ channels. Upon patch excision, a fast rundown of the current was observed. Application of 100 µM polylysine, which sequesters endogenous anionic phospholipids, induced a further rundown of the residual current. Subsequent application of 5 µM diC₈-PIP₂ to the intracellular side of the membrane activated P2X₄ currents (Fig. 7A,B). A quantitative analysis shows a significantly larger P2X₄ current amplitude (1.7-fold increase, p = 0.0059) in the presence of diC₈-PIP₂ (32 ± 6 pA, n = 7) compared with rundown baseline levels (19 ± 4 pA, n = 7).

View larger version (36K): [in this window] [in a new window]   Figure 7. PIP2 activates P2X4 currents in inside-out macropatches excised from Xenopus oocytes. The macropatch pipette solution contained 100 µM ATP. A, Typical P2X4 currents recorded during 1 s voltage ramps from +100 mV to –100 mV. The chelator poly-Lysine (polyK) was applied at 100 µM and the phospholipid diC8-PIP2 (PIP2) was applied at 5 µM. B, Time course of averaged peak currents measured at –80 mV during 5 s intervals.

 
A proximal C-terminal sequence of P2X₄ subunit is critical for phosphoinositide binding
Phosphoinositides are negatively charged lipids so we investigated potential interactions with basic residues on the intracellular domain of the P2X₄ channel subunit, using a lipid strip assay. The proximal region of the C-terminal domain displays a cluster of basic amino acids potentially involved in the interaction with phosphoinositides. A P2X₄ peptide containing the sequence C₃₆₀–V₃₇₅ (Fig. 8A) was expressed as a GST-fusion protein, whereas GST protein alone was used as negative control for the lipid binding assay. No specific binding was observed when the GST protein alone or GST fused with the N-terminal sequence F₁₂–V₂₉ was tested (Fig. 8B). However the GST–P2X₄ (C₃₆₀–V₃₇₅) construct selectively bound to several phosphatidylinositides including PI(3)P, PI(4)P, PI(5)P, PI(3,4)P₂, PI(3,5)P₂, PIP₂, and PIP₃. It also showed strong binding to phosphatidylserine and phosphatidic acid. The neutral lipids phosphatidylethanolamine and phosphatidylcholine did not display any affinity for the GST–P2X₄ (C₃₆₀–V₃₇₅) construct.

View larger version (54K): [in this window] [in a new window]   Figure 8. Direct binding of phosphoinositides to the P2X4 C-terminal domain. A, Primary structure of the proximal C terminus of rat P2X4 subunit showing intracellular lysine and arginine residues candidate for phospholipid binding. B, Specific phospholipid-binding pattern of the GST-tagged 16 aa C-terminal P2X4 peptide C360–V375. The C-terminal peptide binds directly to several biologically active phosphoinositides including PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PIP2, and PIP3 (n = 6).

 

	Discussion

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We demonstrate here a specific modulation of P2X₄ receptor function by phosphoinositides. This modulation is physiologically relevant as both native P2X₄ currents and P2X₄-mediated calcium entry are sensitive to PIP₂ and PIP₃ levels in microglial cells. Microglia are immunocompetent cells that respond to adverse conditions in the CNS, such as neurodegeneration, trauma, ischemia, inflammation, and infection. Increasing evidence indicate that microglia also play a critical role in the pathogenesis of neuropathic pain, a debilitating chronic condition that can occur after peripheral nerve damage. After nerve injury, microglia located in the ipsilateral dorsal horn of spinal cord become activated and upregulate the expression of P2X₄ subunit (Tsuda et al., 2003). Recent findings suggest that ATP-induced activation of microglia evokes the release of BDNF and downregulation of the neuronal cotransporter KCC2, leading to postsynaptic changes in the electrochemical gradient of chloride ions and disinhibition (Coull et al., 2003, 2005). BV-2 cells have been established as a model of murine microglia. Native microglial cultures have serious limitations including short survival time ex vivo, heterogeneity in activation states (Lawson et al., 1990; Ren et al., 1999) and the process of isolating primary microglia can result in phenotypical changes (Frank et al., 2006). P2X₄ and P2X₇ are the major P2X subtypes expressed in BV-2 cells (Raouf et al., 2007). We successfully isolated P2X₄ currents from P2X₇ currents by using a dual pharmacological strategy based on the higher sensitivity of mouse P2X₄ to ATP compared with that of mouse P2X₇ (Townsend-Nicholson et al., 1999; Raouf et al., 2007) and on the use of ivermectin, a selective allosteric potentiator of P2X₄-containing receptors (Khakh et al., 1999; Toulmé et al., 2006). Our patch-clamp recordings data showed reduced P2X₄ current density after blockade of PI3K and PI4K with wortmannin at 100 nM and 35 µM, respectively. The ATP-induced microglial release of the proalgesic cytokines TNF- and interleukin-6 involve Ca²⁺-dependent pathways (Hide et al., 2000; Shigemoto-Mogami et al., 2001; Hanisch, 2002). P2X₄ receptor channels are highly permeable to Ca²⁺ ions (Egan and Khakh, 2004) and we used this property to measure in ratiometric imaging a significant reduction of ATP-induced ivermectin-sensitive Ca²⁺ ion entry after treatment with wortmannin. Altogether, our results show that both PIP₂ and PIP₃ levels regulate the activity of native P2X₄ receptor channels in BV-2 microglial cells. It is possible that a large Ca²⁺ influx through P2X₄ receptor channels could activate Ca²⁺-dependent isoforms of phospholipase C, leading to decreased PIP₂ levels and subsequent decreased channel responses. Interestingly, such a mechanism of Ca²⁺-induced inactivation of PIP₂-sensitive channels through activation of phospholipase C has been described recently for TRP channels (Rohács et al., 2005; Thyagarajan et al., 2008).

The modulation of P2X₄ responses by phosphoinositides does not depend on a cell-specific context since we reconstituted it in the Xenopus oocyte expression system. Wortmannin-induced phosphoinositides depletion in oocytes led to reduced maximal P2X₄ currents, lower sensitivity to ATP, longer recovery times following repeated agonist applications, as well as slower current activation and inactivation kinetics. We conclude that ATP becomes a partial agonist for P2X₄ when PIP₂ and PIP₃ levels are depleted, i.e., phosphoinositides are required cofactors for the full expression of P2X₄ function. Treatment with wortmannin at micromolar concentrations could potentially affect many intracellular pathways. However we validated the selective blockade of phosphoinositides synthesis by normalizing P2X₄ current amplitude as well as kinetics with cytoplasmic injections of PIP₂ or PIP₃. Interestingly, depleting PIP₃ levels has an impact on the amplitude of P2X₄ currents and on their recovery time, but not on their kinetics of activation or inactivation which are sensitive to PIP₂ only. Therefore P2X₄ channels are sensitive to both PIP₂ and PIP₃ albeit through different mechanisms of regulation.

Although earlier studies conducted on the modulation of ion channels by phosphoinositides showed a predominant role of PIP₂ as a signaling molecule (Suh and Hille, 2008), there is increasing evidence that PIP₃ also regulates various ion channels including K⁺ inward rectifier channels (Rohács et al., 2003), Ca²⁺-activated K⁺ channels (Srivastava et al., 2005), epithelial Na⁺ channels (Pochynyuk et al., 2006) as well as cyclic nucleotide-gated channels (Zhainazarov et al., 2004). Among the P2X receptors, the desensitization of the P2X₂ subtype is modulated by PI(3)P and PI(3,5)P₂ (Fujiwara and Kubo, 2006) and we have recently reported that the P2X₁ and P2X₇ subtypes are sensitive to PIP₂, but not to PIP₃ (Zhao et al., 2007; Bernier et al., 2008). Therefore the sensitivity of P2X₄ receptor channels to both PIP₂ and PIP₃ indicates that phosphoinositides regulate P2X receptor channel function in a subunit-specific manner.

We also provide evidence that modulation of P2X₄ channels results from the direct effects of protein-lipid interactions on channel function, and not from indirect effects on trafficking and endocytosis since PIP₂ is able to rescue P2X₄ currents from rundown in inside-out macropatches excised from oocytes. P2X₄ channel activity was stimulated by direct application of PIP₂ to the intracellular side of the macropatch, likely by promoting the exit of P2X₄ channels from their desensitized state. This could explain the longer recovery time and the slower kinetics of P2X₄ currents recorded in whole cell configuration when PIP₂ levels are low after treatment with wortmannin. Another strong argument for the direct effect of phospholipids on P2X₄ function is based on the fact that PIP₂ and PIP₃, as well as other phosphoinositides, are able to interact directly with the proximal C-terminal subdomain Cys₃₆₀–Val₃₇₅ of P2X₄ subunits in vitro. This region contains a cluster of basic residues at the right location for interacting with membrane-bound anionic phospholipids, and it shows significant conservation among all known P2X subunits. By analogy with other channels (Suh and Hille, 2008), the highly negatively charged head group in phosphoinositides is thought to interact directly with a distinct positively charged binding site in this region of the P2X₄ subunit. The exact contribution of each of the 7 candidate residues (6 lysines and 1 arginine, see sequence in Fig. 8) to the binding of PIP₂ and/or PIP₃ will remain to be investigated.

The fact that the N-terminal domain of P2X₄ did not bind to any of the phosphoinositides tested confirms an important regulatory role for the C-terminal domain that also contains determinants of desensitization (Fountain and North, 2006) and internalization (Royle et al., 2005). Our results in macropatch recordings and in lipid binding assay strongly suggest that direct interactions between P2X₄ channels and phosphoinositides mediate the modulation observed in microglia as well as in Xenopus oocytes.

The relevance of the modulation of microglial and neuronal P2X₄ function by PIP₂ and/or PIP₃ in pathophysiological conditions like neuropathic pain will remain to be established. However, in a broader context, our results highlight a positive and subunit-specific modulation of P2X receptor channels by phosphoinositides through direct channel–lipid interactions. Many metabotropic receptors regulate phosphoinositide levels through the activation of PI3K or phospholipase C, therefore we will need to integrate their exact role on P2X function in our understanding of ionotropic ATP signaling in the nervous system and in peripheral tissues.

	Footnotes

 
Received July 1, 2008; revised Oct. 10, 2008; accepted Oct. 14, 2008.

This work has been supported by operating grants from Canadian Institutes of Health Research (CIHR) (P.S.), Agence Nationale pour la Recherche and Projet International de Coopération Scientifique du Centre National de la Recherche Scientifique (E.B.G.), and National Institutes of Health (D.L.). L.P.B. holds a studentship from the CIHR training grant "Pain: Molecules to Community."

Correspondence should be addressed to Dr. Philippe Séguéla, Montreal Neurological Institute, 3801 University, Suite 778, Montréal, Québec, Canada H3A 2B4. Email: philippe.seguela{at}mcgill.ca

Copyright © 2008 Society for Neuroscience 0270-6474/08/2812938-08$15.00/0

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