Phosphoinositides regulate P2X4 ATP-gated channels through direct interactions

Abstract

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X(4) 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 P2X(4) function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P(2) (PIP(2)) and PI(3,4,5)P(3) (PIP(3)), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X(4) receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP(2) and PIP(3) with wortmannin significantly decreased P2X(4) current amplitude and P2X(4)-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 P2X(4) 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 PIP(2) on excised inside-out macropatches rescued P2X(4) currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X(4) subunit (Cys(360)-Val(375)) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP(2) and PIP(3) on P2X(4) function through direct channel-lipid interactions.

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.

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 Δ 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.

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).

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.

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).

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).

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.

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).