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Functional Properties of Internalization-Deficient P2X₄ Receptors Reveal a Novel Mechanism of Ligand-Gated Channel Facilitation by Ivermectin

Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5543, Université Victor Segalen Bordeaux 2, Bordeaux, France (E.T., M.G., E.B.-G.); and the Max-Planck Institute for Experimental Medicine, Göttingen, Germany (F.S.)

Received September 12, 2005; accepted November 10, 2005

	Abstract

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Although P2X receptors within the central nervous system mediate excitatory ATP synaptic transmission, the identity of central ATP-gated channels has not yet been elucidated. P2X₄, the most widely expressed subunit in the brain, was previously shown to undergo clathrin-dependent constitutive internalization by direct interaction between activator protein (AP)2 adaptors and a tyrosine-based sorting signal specifically present in the cytosolic C-terminal tail of mammalian P2X₄ sequences. In this study, we first used internalization-deficient P2X₄ receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X₄ channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X₄ receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X₄, did not potentiate the maximal current of internalization-deficient rat or human P2X₄ receptors. We demonstrated that binding of ivermectin onto wild-type P2X₄ channels increased the fraction of plasma membrane P2X₄ receptors, whereas surface expression of internalization-deficient P2X₄ receptors remained unchanged. Disruption of the clathrin-mediated endocytosis with the dominant-negative mutants Eps15 or AP-50 abolished the ivermectin potentiation of wild-type P2X₄ channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic 7 acetylcholine (n7ACh) receptors and the potentiation of acetylcholine current by ivermectin was suppressed when the same dominant-negative mutants were expressed. These data showed that potentiation by ivermectin of both P2X₄ and n7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.

There is currently accumulating evidence that ligand-gated channels from the "cys loop"- or glutamate-receptor family interact with many intracellular proteins implicated in the regulation of the number, subcellular localization, and anchoring of receptors (Moss and Smart, 2001; Mayer, 2005). In addition, receptor-associated proteins have been shown to modulate functional properties of ligand-gated channels. Microtubule-associated protein MAP1B (Billups et al., 2000) or GABA receptor-associated protein (Chen et al., 2000) modulate the agonist sensitivity and/or kinetics of GABA-gated channels, whereas postsynaptic density 95 accelerates the rate of recovery from desensitization of GluR6 (Bowie et al., 2003). In comparison, very little is known about neuronal P2X receptor-associated proteins. Recent studies on trafficking of P2X receptors demonstrated convincingly that P2X₄ receptors cycle into and out of the plasma membrane, providing a means of modulating receptor density (Bobanovic et al., 2002). Other ligand-gated channels such as GABA_A or AMPA receptors undergo regulated cycling. This has been shown to modulate excitatory and inhibitory transmission (Kittler et al., 2000; Carroll et al., 2001). Rat P2X₄ receptors interact directly with µ2 (also called AP-50) subunits of AP2 adaptor complex through a novel tyrosine-based sorting signal at the C-terminal tail of P2X₄ subunits (Royle et al., 2002, 2005), which ensures the recruitment and selection of P2X₄ receptors for their constitutive- and agonist-dependent internalization (Bobanovic et al., 2002; Royle et al., 2002). This YxxGL motif is totally conserved among mammalian P2X₄ sequences but is absent from other P2X subtypes. Mutation of the binding site for AP2 on P2X₄ causes a decrease in internalization and a resulting dramatic increase in the surface expression of P2X₄ receptors (Royle et al., 2005). In this study, we examined whether interaction with AP2 influenced the functional properties of P2X₄ channels using internalization-deficient rat or human P2X₄ channels. Disruption of AP2/P2X₄ interaction produced unique P2X₄ receptor functional properties consistent with those of native P2X responses. Furthermore, in the absence of interaction with AP2, P2X₄ channels were found to be insensitive to ivermectin. In this study, we demonstrated that the binding of ivermectin onto receptors increased the number of surface P2X₄ channels (as well as nicotinic 7 acetylcholine receptors) and that this regulatory effect of ivermectin could be disrupted by interfering with clathrin/AP2-mediated endocytosis process.

	Materials and Methods

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Molecular Biology. Wild-type rat P2X₄ and rat 7 nicotinic acetylcholine (kindly provided by Dr. P. Séguéla, Montreal Neurological Institute, Montreal, QC, Canada) plasmids were used in this work, but some experiments were reproduced with wild-type or mutated human P2X₄ receptors (specified in the text). Receptor mutants were generated by polymerase chain reaction with the wild-type plasmid as a DNA template using PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) to minimize artifactual mutations. Point mutations were constructed using the Quickchange site-directed mutagenesis system (Stratagene). For construction of C-terminal-truncated rat P2X₄ receptors (P2X₄ 377), we used a 3' primer derived from the amino acid sequence R³⁶⁸DK KYKYVED³⁷⁷ of P2X₄ followed by an artificial stop codon and a terminal XbaI site. Tagged-P2X₄ and -7 subunits were generated as reported previously for other subunits (Boué-Grabot et al., 2004a). Plasmids encoding the dominant-negative Eps15 mutant (Eps 15 DN or GFP-ED95/295) and a control construct (Eps15 control, also called GFP-DIIID2; Benmerah et al., 1996) were provided by Dr. Agnès Hémar (Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5091, Bordeaux, France). The cDNA coding for rat µ2 (or AP-50) was generated with RNA extracted from the brain by reverse transcription-polymerase chain reaction. The dominant-negative AP-50 subunit was made by sequentially mutating D¹⁷⁶A and W⁴²¹A as described previously (Nesterov et al., 1999; Royle et al., 2002). All constructs were subcloned into pcDNA₃ (Invitrogen, Carlsbad, CA) and verified by automatic dideoxy DNA sequencing (MWG Biotech, High Point, NC).

Xenopus laevis Oocyte Injection and Electrophysiology. Oocytes were prepared as described previously (Boué-Grabot et al., 2004a). Stage V and VI oocytes were manually defolliculated before nuclear injection (Nanoject II; Drummond Scientific, Broomall, PA) of cDNAs. The oocytes were then incubated in Barth's solution containing 1.8 mM CaCl₂ and gentamycin (10 µg/ml; Sigma-Aldrich, St. Louis, MO) at 19°C for 1 to 4 days before electrophysiological recordings. Two-electrode voltage-clamp recordings were carried out at room temperature using glass pipettes (1–2 M) filled with 3 M KCl solution to ensure a reliable holding potential. Oocytes were voltage clamped at –60 mV, and the membrane currents were recorded through an OC-725B amplifier (Warner Instruments, Hamden, CT) and digitized at 500 Hz. Oocytes were perfused at a flow rate of 10 to 12 ml/min with Ringer's solution (Na⁺-R), pH 7.4, containing 115 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 1.8 mM (or 0.1 mM) CaCl₂, and 10 mM HEPES or using an extracellular solution (NMDG⁺-R) containing 5 mM NaCl, 110 mM NMDG, 5 mM NaOH, 2.5 mM KCl, 0.1 mM CaCl₂, and 10 mM HEPES (Boué-Grabot et al., 2004b). Agonists and drugs (purchased from Sigma-Aldrich) were prepared at their final concentration in the perfusion solution and applied using a computer-driven valve system (Ala Scientific, Westbury, NY). For all experiments, ivermectin was initially dissolved (10 mM) in DMSO and kept at –20°C for up to 2 weeks. The concentration of DMSO in the final solution was 0.03% (P2X₄ experiments) and 0.3% (7 experiments). Application of the vehicle alone did not affect ATP or ACh responses.

Data Analysis. All data were analyzed using Prism 4.0 (GraphPad Software Inc., San Diego, CA). Numerical values are expressed as mean ± S.E.M. from n determination. Current-voltage relations were obtained by applying 200-ms voltage ramps from –80 to +50 mV. Reversal potentials (E_REV) were estimated from the I–V relationship, determined by linear regression test analysis. The ratio of NMDG⁺ to Na⁺ permeability (P) was calculated using the function P_NMDG⁺/P_Na⁺ = exp(E_REVF/RT) as described previously (Khakh et al., 1999a). E_REV is the observed shift in reversal potential, F is the Faraday constant, R is the gas constant, and T is absolute temperature. Dose-response curves were fitted to the Hill sigmoidal equation, and EC₅₀ values were determined by nonlinear regression analysis. Statistical comparisons were assessed using Student's t test. The differences were considered significant if p < 0.05.

Surface Biotinylation and Immunoblotting. Biotinylation experiments were performed essentially as described previously (Ennion and Evans, 2002). To examine the effect of ivermectin on surface receptor expression, 25 to 30 oocytes were incubated in Ringer's solution containing 0.5 mg/ml sulfo-N-hydroxysuccinimide-biotin (Pierce, Rockford, IL) and either ivermectin or DMSO (control) for 2 min at room temperature. Then, oocytes were immediately placed in a chilled Ringer-biotin solution for 30 min at 4°C with gentle agitation. After washing four times in Ringer's solution, oocytes were homogenized in a volume of 20 µl/oocyte of buffer H (10 mM HEPES, 0.3 M sucrose, protease inhibitor cocktail; Roche Diagnostics, Indianapolis, IN), and solubilized with 1% Triton X-100 at 4°C for 2 h. After centrifugation (4°C) at 10,000g for 10 min, 7.5 to 15 µl of the supernatant was stored at –20°C before SDS-PAGE to assess total receptor fraction. The remaining supernatant was incubated at 4°C for 4 h with 40 µl of Immunopure Immobilized Neutravidin binding proteins (Pierce) to precipitate surface proteins. Beads were washed four times with buffer H, and adsorbed proteins were eluted with 1 volume of SDS sample buffer at room temperature. Surface and total proteins were separated on SDS-PAGE and revealed by Western blotting using either anti-P2X₄ receptor antibodies at a 1:4000 dilution (0.25 µg/ml; Nicke et al., 2005) or anti-GFP antibodies at a 1:8000 dilution (Invitrogen). Quantification of Western blots was performed using Image J 1.33u (http://rsb.info.nih.gov/nih-image/Default.html) software, and surface-total protein ratio was related to the number of oocytes (on average, total protein bands corresponded to 0.37 to 0.75 oocytes, and surface signals corresponded to 20 oocytes).

	Results

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Mutation of the Endocytosis Motif of P2X₄ Receptors Increases Functional Receptors in Oocytes. Royle et al. (2002, 2005) identified a C-terminal P2X₄ motif (Y³⁷⁸xxGL³⁸²) of interaction with AP-50 (also called µ2) protein of the AP2 complex required for the constitutive endocytosis of P2X₄ receptors. This motif is conserved among the mammalian P2X₄ sequences but is absent in other P2X subtypes. Mutation of this sorting signal increased the surface expression of P2X₄ receptors 5- to 8-fold in transfected neurons (Royle et al., 2002). We eliminated the site of interaction with AP2 of rat P2X₄ subunit sequence either by truncating at residue 377 (P2X₄ 377) or replacing tyrosine 378 by an alanine (P2X₄ Y378A). Two days after the injection of 1.5 ng of cDNAs encoding wild-type P2X₄, P2X₄ 377, or P2X₄ Y378A in X. laevis oocytes, ATP-evoked inward currents recorded by two-electrode voltage clamp displayed moderately desensitizing kinetics for all channels tested (Fig. 1A). However, the desensitization rate of truncated P2X₄ receptors was more variable than for wild-type or P2X₄ Y378A receptors; 24 to 63% of the oocytes (four different batches) expressing truncated P2X₄ receptors displayed a slowly desensitizing phenotype (see Fig. 1A, inset). Variation of phenotype was previously reported with C-terminal-truncated P2X₂ receptor (Boué-Grabot et al., 2000). The amplitude of ATP current was, respectively, 0.27 ± 0.06 µA (n = 20), 7.44 ± 0.45 µA (n = 21), and 7.41 ± 0.66 µA (n = 9) for P2X₄ WT, P2X₄ 377, and P2X₄ Y378A (Fig. 1A), revealing a large (25-fold) increase in the functional response of mutated P2X₄ subunits. A 10-fold increase in current amplitude was similarly observed when oocytes injected with 1.5 ng of cDNAs coding for human P2X₄ Y378A (I_ATP = 12.94 ± 2.1 µA, n = 8) or human P2X₄ WT (I_ATP = 1.28 ± 0.48 µA, n = 9) were compared. The enhancement of maximal current was dependent on the amount of DNA injected (Fig. 1B) but was independent of the time course of channel expression (Fig. 1C). Data in Fig. 1B indicated that 30 times less DNA was necessary to reach maximal current of P2X₄ 377 versus P2X₄ WT. According to previous data that demonstrated that disruption of interaction with AP2, inferred by mutated P2X₄ receptors, increased the number of surface P2X₄ receptors in transfected HEK cells or neurons (Bobanovic et al., 2002; Royle et al., 2002), the observed increases in maximal current are probably related to a higher surface expression of mutated P2X₄ receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Mutation of the endocytosis motif of P2X4 receptors increases ATP current density. A, representative currents from oocytes expressing rat P2X4 WT, P2X4 377, or P2X4 Y378A receptors in response to 5-s application of 100 µM ATP. Deletion (P2X4 377) or mutation (P2X4Y378A) of P2X4 receptor endocytosis motif significantly increased the amplitude of current in comparison with P2X4 WT receptors (***, p < 0.001). Recordings were performed the same day (2 days after injection of 1.5 ng of each cDNA). Note in the inset the slow desensitizing kinetics observed in 24 to 63% oocytes expressing P2X4 377. B, amplitude of 100 µM ATP currents recorded 2 days after nuclear injection in X. laevis oocytes of different quantities of cDNAs encoding P2X4 WT () or P2X4 377 (). Mean peak currents were normalized to the response of oocytes injected with 1.5 ng of P2X4 WT cDNA; n = 4 and 20. C, time course of P2X4 WT () or P2X4 377 currents () respectively normalized to the mean of currents recorded at day 2 (n = 7–13). Holding potential was –60 mV.

Functional Characterization of Endocytosis-Deficient P2X₄ Receptors. ATP-induced currents in oocytes expressing wild-type or mutant P2X₄ channels fully recovered after repeated applications at 4-min intervals (not shown). High density of P2X₂ channels was shown to alter channel properties in oocytes (Fujiwara and Kubo, 2004). To analyze functional properties of internalization-deficient P2X₄ receptors and rule out the possibility of density-dependent changes, wild-type and mutant rat P2X₄ receptors were expressed at similar low levels by injecting, respectively, 1.5 and 0.1 ng of cDNA encoding subunits. Unless specified, the following experiments were performed under these conditions. The current-voltage relationship was determined by measuring the peak of ATP (100 µM) response obtained at several membrane potentials (from –60 to 20 mV, n ranged from 2 to 5) and normalized to the current obtained at –60 mV. Superimposed I–V curves for WT and mutated P2X₄ channels with reversion potential of–8.1 ± 6.6 and –9.0 ± 7.2 mV, respectively, showed that mutation did not change the cation selective permeability of P2X₄ channels (Fig. 2A). Concentration-response curves for oocytes expressing either P2X₄ or P2X₄ Y378A at a similar current density (I_ATP < 6 µA at saturating concentration) were obtained by recording activity of both channels at different concentrations of ATP. EC₅₀ of ATP was found to be 0.29 ± 0.15 µM and 4.32 ± 1.4 µM, respectively, for P2X₄ Y378A and wild-type P2X₄ channels (Fig. 2B; n ranged from 4 to 12), indicating that P2X₄ Y378A receptors were 15 times more sensitive to ATP than wild-type receptors. Rat P2X₄ channels are typically considered insensitive to -methylene-ATP, although a slight effect at concentrations higher than 100 µM has been reported (Khakh et al., 1999b; Jones et al., 2000; North, 2002). As illustrated in Fig. 2C, -methylene-ATP evoked measurable currents only at concentrations superior to 100 µM for wild-type P2X₄, representing 15% of 100 µM ATP currents. Similar results were observed for P2X₄ Y378A channels (I_ATP = 6.7 ± 0.7 µA, n = 12; Fig. 2C). It is noteworthy that at a higher density of P2X₄ Y378A channels (I_ATP = 13 ± 0.8 µA, n = 11), -methylene-ATP evoked robust responses, representing 40% of ATP currents. The dose-response curve displayed an EC₅₀ of 54 ± 3 µM, demonstrating that -methylene-ATP was a potent agonist of P2X₄ Y378A channels depending on the channel density (Fig. 2C).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Functional properties of endocytosis-deficient P2X4 channels expressed in X. laevis oocytes. A, current-voltage relationships of wild-type P2X4 () or P2X4 Y378A () receptors. Currents evoked by 100 µM ATP were normalized to the current recorded at a holding potential of –60 mV. B, ATP dose-response curves of P2X4 WT (, n = 4–12) and P2X4 Y378A (, n = 4–12) receptors normalized to 100 µM ATP-induced current. Current density was similar for both channels. C, concentration-effect curves for -methylene-ATP at low density (IATP < 7 µA, and , respectively, for WT- and mutated-P2X4) or at high density (, IATP > 10 µA for P2X4 Y378A receptors). -Methylene-ATP-induced currents were normalized to 100 µM ATP responses. Insets, superimposition of representative currents evoked either by ATP or -methylene-ATP (100 µM each) for oocytes expressing P2X4 WT at low density or P2X4 Y378A receptors at high density. D, inhibition of 10 µM ATP currents induced by coapplication of suramin (30 or 100 µM) or 5-min preincubation of 100 µM suramin (n = 2–5). E, mean peak currents evoked by 10 µM ATP at pH 6.4, 7.4, or 8.4 or by 10 µM ATP at pH 7.4 in the presence of 10 µM Zn2+. Currents are normalized to the value obtained at pH 7.4 in the same cell (n = 5–7).

Suramin is an antagonist of P2X receptors with low efficacy on P2X₄ receptors (North, 2002; Fig. 2D). Coapplication or preincubation (5 min) of the antagonist (tested at 30 and 100 µM) reduced ATP-induced response by a maximum of 33.8 ± 0.6% or 25.9 ± 6.6% (n = 2–5, p > 0.05) respectively for wild-type and mutant P2X₄ channels, indicating that mutation produced no change in suramin efficacy. Moreover, as summarized in Fig. 2E, mutation of the endocytic motif did not modify neither the well established potentiation by Zn²⁺ (10 µM; Soto et al., 1996) nor the pH dependence of the action of ATP on P2X₄ channels (North, 2002).

Dilation Properties of Endocytosis-Deficient P2X₄ Receptors. P2X₄ channels undergo permeability changes during prolonged or repetitive activation (Khakh et al., 1999a; Virginio et al., 1999). As reported and illustrated in Fig. 3, A and B, application of ATP (100 µM) in low-Ca²⁺ Ringer's solution opens in milliseconds a channel permeable to small cations (state I₁) and, then, in the continuous presence of ATP, the channel becomes permeable to much larger cations (state I₂). I₂ represented 241.8 ± 77.22% (n = 6) of I₁ for wild-type P2X₄ receptors (Fig. 3D). We tested whether P2X₄ Y378A channels underwent permeability changes. We found that prolonged application (2 min) of ATP evoked biphasic P2X₄ Y378A currents. Contrary to P2X₄WT, I₂ represented only 35.1 ± 10.2% (n = 7) of I₁ for P2X₄ Y378A channels (Fig. 3, A, B, and D). We determined the reversal potential of ATP-evoked current as a function of the time either in Na⁺-R solution or with NMDG⁺ replacing all the extracellular Na⁺ (Fig. 3C). The reversal potentials for both channels in Na⁺-R solution of I₁ were constant and close to 0 mV (Fig. 3E). The reversal potential of P2X₄ WT in NMDG⁺ solution shifted by 22 mV from –59.9 ± 7.3 mV (state I₁) to –37.5 ± 1.9 mV (state I₂) during ATP application in NMDG⁺-R solution, showing the progressive permeability increase of P2X₄ WT channel pore (Fig. 3E). The reversal potential of I₁ current for P2X₄ Y378A was –21.2 ± 0.7 mV and then slightly decreased to –24 ± 2 mV at the entry in I₂ state (0.5 min in ATP; Fig. 3C). These results showed that P2X₄ Y378A receptors are immediately permeable to NMDG⁺, suggesting that cytosolic domains played an important role in permeability change (Fisher et al., 2004). Although no significant increase in NMDG⁺ permeability occurred during prolonged ATP application (Fig. 3E), P2X₄ Y378A displayed biphasic responses showing that transition between I₁ and I₂ states is a complex mechanism that not only reflects changes in permeability.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Permeability changes of endocytosis-deficient P2X4 receptors. A, representative biphasic currents (I1 and I2) elicited by prolonged application of 100 µM ATP in Ringer's solution containing 0.1 mM Ca2+ from oocytes expressing P2X4 WT or P2X4 Y378A receptors at low-current density. The first peak (I1) develops rapidly and desensitizes; the second peak (I2) develops slowly over several minutes. B, mean amplitudes of I1 and I2 currents (microamperes) from experiments described in A. D, I2/I1 ratio is significantly decreased for P2X4 Y378A receptors (n = 7) compared with P2X4 WT receptors (n = 6); *, p < 0.05. C, time course of reversal potential change in NMDG+ Ringer's solution. Each point represented the mean ± S.E.M. (n = 4–6) of the reversal potential obtained from voltage ramps (–80 to + 50 mV; 200 ms). E, table summarizing reversal potentials, shifts in reversal potentials, and the relative permeability PNMDG+/PNa+ of P2X4 WT or P2X4 Y378A I1 and I2 conducting states in NMDG+ or Na+ Ringer's solution.

Altogether, these results show that endocytosis motif is critical for P2X₄ function. These differences, observed at similar current densities, eliminated the possibility of density-dependent changes and suggested that loss of interaction with AP2 proteins modulates function of P2X₄ receptors.

Ivermectin Does Not Potentiate Endocytosis-Deficient P2X₄ Receptors. Ivermectin slows deactivation and increases maximum ATP currents through rat and human P2X₄ receptors but not through other P2X receptor subtypes (Khakh et al., 1999b; Priel and Silberberg, 2004). Figure 4, A and B, shows that 2-min application of ivermectin (3 µM) significantly increased ATP currents from oocytes expressing rat or human P2X₄ channels. However, we noticed that ivermectin potentiated ATP currents with an initial amplitude of less than 2 µA but had no significant effect on oocytes showing higher channel density (I_ATP > 2 µA; Fig. 4B, n = 37). This finding has not been reported in previous studies using both P2X₄ orthologs (Khakh et al., 1999b; Priel and Silberberg, 2004). In agreement with previous work (Khakh et al., 1999b), ivermectin increased the amplitude of initial ATP current (I < 2 µA; Fig. 4E) by 297 ± 21% or 287 ± 64% for rat or human P2X₄ WT channels, respectively. In contrast, ivermectin (3 or 10 µM) failed to increase the peak current of rat or human P2X₄ Y378A and rat P2X₄ 377 at all current densities tested (I_ATP ranged from 0.1 to 10 µA, n = 53; Fig. 4, C and D). After preincubation with 3 µM ivermectin, ATP current represented 141 ± 18% (n = 11), 76 ± 11% (n = 6), or 133 ± 17% (n = 10) of the initial ATP responses, respectively, for rat, human P2X₄ Y378A, or rat P2X₄ 377 (to compare wild-type and mutant P2X₄, only I_ATP < 2 µA were taken into account). However, it is important to note that the deactivation rate of wild-type and both mutated channels was slowed by ivermectin (Fig. 4, A and C). These observations, in agreement with previous studies (Khakh et al., 1999b; Priel and Silberberg, 2004), showed that the maximal current increase and the deactivation slowdown induced by ivermectin result from distinct mechanisms on mammalian P2X₄ channels. The surprising result showing that endocytosis motif was required for the peak current potentiation by ivermectin (Fig. 4) and the intriguing fact that functional properties of internalization-deficient P2X₄ receptor mutants (Figs. 2 and 3) were similar to those observed for wild-type P2X₄ receptors in the presence of ivermectin (Khakh et al., 1999) leads us to hypothesize that ivermectin caused potentiation by increasing the number of surface receptors.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Ivermectin does not potentiate mutated rat or human P2X4 receptor peak current. A and C, representative currents elicited by ATP (100 µM) before and after 2-min preincubation with 3 µM ivermectin (IVM). A, 2-min preapplication of IVM (3 µM) produced a large increase in rat P2X4 WT channel currents induced by 100 µM ATP and slowed deactivation (arrows). B, potentiation by IVM (3 µM) of rat or human P2X4 WT peak current (gray panel) occurred when initial ATP current was inferior to 2 µA (n = 24). No potentiation was observed for higher initial ATP current (I > µA, n = 13). C, IVM produced no significant increase in ATP peak current of rat P2X4 Y378A receptors but slowed the rate of deactivation of P2X4 Y378A channels (arrows). D, IVM induced no significant effect on ATP currents whatever the amplitude of initial ATP responses for rat or human P2X4 Y378A and rat P2X4 377 receptors. E, means of potentiation by IVM. Only oocytes with initial ATP responses < to 2 µA were taken in account.

The Functional Potentiation of P2X₄ Receptors by Ivermectin Is Accompanied by an Increase in Receptor Surface Expression. To directly test whether an increase in the number of surface receptors could underlie the ivermectin effect on P2X₄ channels, we examined the relative amount of surface-specific protein expression by conducting surface biotinylation experiments on oocytes expressing P2X₄ receptors. Detection of total and surface P2X₄ subunits using a C-terminal P2X₄-specific antibody (Nicke et al., 2005) revealed the weak surface expression of P2X₄ receptors (Fig. 5A). Surface fraction represented 0.3% of total P2X₄ proteins under control conditions (Fig. 5A, lane C). In the presence of 3 µM ivermectin (2- or 30-min incubation), we observed a significant increase in surface signals, whereas total P2X₄ expression remained unchanged (Fig. 5, A and D). Comparison of the relative surface/total ratio in the absence or the presence of ivermectin indicated that ivermectin increased plasma membrane P2X₄ subunits by 300 to 450% (for 30- or 2-min incubation, respectively, four independent experiments). The increase induced by ivermectin was not attributable to the presence of DMSO because control oocytes were incubated in Ringer's solution containing 0.03% DMSO. The absence of signal from noninjected oocytes (Fig. 5A, lanes NI) or oocytes expressing either P2X₄ 377 or P2X₄ Y378A (Fig. 5B) demonstrated the specific detection of wild-type P2X₄ and suggested that the residue Y³⁷⁸ is critical for the C-terminal antigenicity. Contrary to P2X₄ WT, we showed, using anti-GFP antibodies, that similar ivermectin treatment had no effect on the relative surface fraction of P2X₄ Y378A-yellow fluorescent protein receptors (Fig. 5, C and D). These results provide evidence that the 3-fold potentiation of P2X₄ currents caused by ivermectin can be accounted for by an increase in the number of surface receptors.

View larger version (43K): [in this window] [in a new window]   Fig. 5. Surface expression of P2X4 WT receptors is increased by ivermectin. A, P2X4 WT expressing or noninjected (NI) oocytes were incubated in ivermectin (3 µM) or in vehicle (c) 2- or 30-min before surface biotinylation. Representative Western blot shows P2X4 immunoreactivity in total or surface biotinylated extracts from oocytes expressing P2X4 WT, whereas no signal is detected from noninjected oocytes (NI). B, anti-P2X4 antibodies directed against the C-terminal of P2X4 receptors did not detect P2X4 377 or P2X4 Y378A receptors in total protein extracts from oocytes in which functional expression was assessed by voltage-clamp recordings. C, anti-GFP Western blot on total extracts or biotinylated surface proteins from oocytes expressing P2X4 Y378A-yellow fluorescent proteins. Oocytes were incubated in vehicle (c) or ivermectin (3 µM) 2 min before biotinylation. D, means of surface/total expression ratio from experiments described in A and C show that IVM (+) induced a dramatic increase in surface P2X4 fraction, whereas surface expression of mutated P2X4 receptors was unchanged.

Up-Regulation by Ivermectin Is Driven by Endocytosis. The rapid time course of the ivermectin-induced increase of P2X₄ receptors expression on the plasma membrane limits the mechanism through which it could occur (e.g., trafficking rather than receptor synthesis or assembly). To test whether the effect of ivermectin on wild-type P2X₄ channels involved AP2/clathrin-dependent endocytosis, we disrupted the clathrin-mediated pathway using well characterized dominant-negative mutants of endocytosis. We first coinjected oocytes with a mixture (1:1.5 ratio) of cDNAs encoding wild-type P2X₄ receptors and either the dominant-negative mutant of Eps15 (Eps15 DN) or a control Eps15 construct (Benmerah et al., 1996). The amplitude of ATP currents for P2X₄ receptors was significantly higher in the presence of Eps15 DN (I_ATP = 0.16 ± 0.03 µA, n = 7) compared with the Eps15 control (I_ATP = 0.07 ± 0.02 µA, n = 7), showing that Eps15 DN prevented the rapid endocytosis of P2X₄ receptors (Fig. 6, A, B, and E). Moreover, the potentiation of ATP current by ivermectin was significantly decreased in the presence of Eps15 DN (I_ATP after ivermectin represented 168.5 ± 26% of I_ATP control, n = 7), whereas P2X₄ potentiation is unaffected by the presence of Eps15 control (I_ATP after ivermectin represented 347.1 ± 22.5%, n = 14 of I_ATP control; Fig. 6, A, B, and D).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Potentiation of P2X4 WT peak current is dependent on clathrin-dependent endocytosis. A, B, and C, representative 100 µM ATP currents before and after ivermectin preincubation from oocytes coexpressing P2X4 WT receptors with either the dominant-negative mutant of Eps15 (A), a control form of Eps15 (B), or the dominant-negative mutant of AP-50 (C). D, means of amplitude of initial ATP responses showed that expression of P2X4 WT receptors was significantly higher in the presence of Eps15 DN or AP-50 DN than in the presence of their respective protein control (*, p < 0.05; ***, p < 0.0005). E, histograms showing the effect of ivermectin on normalized ATP-induced currents in oocytes expressing P2X4 WT channels plus control or DN Eps15 as well as wild-type or DN AP-50. The expression of both dominant-negative of clathrin-mediated endocytosis significantly decreased the potentiation of P2X4 WT currents by IVM (***, p < 0.0005). ND, not determined.

Ivermectin Increases the Number of Cell Surface n7ACh Receptors by a Mechanism Involving Endocytosis. Preapplication of ivermectin enhances reversibly ACh-evoked current of nicotinic 7 acetylcholine receptors (n7AChR) expressed in X. laevis oocytes and HEK cells (Krause et al., 1998). In addition, ivermectin increases the apparent affinity and cooperativity of the ACh dose-response curve (Krause et al., 1998). This prompted us to investigate ivermectin action mechanism on n7ACh receptors. We expressed rat n7AChR in X. laevis oocytes and showed that 20-s preapplication of ivermectin (30 µM) induced a large and reversible increase (219 ± 13%, n = 13; Fig. 7, A and B) of current evoked by application of ACh (300 µM). To explore further the mechanism of potentiation, we examined the functional consequences of the disruption of clathrin-dependent pathway using the dominant-negative mutants mentioned above. In oocytes coexpressing n7ACh receptors and the dominant-negative Eps15 mutant, ivermectin potentiation was significantly reduced (152.7 ± 9%, n = 18). On the contrary, ivermectin was still able to potentiate n7AChR current evoked by ACh in the presence of Eps15 control (223.9 ± 22.6%; Fig. 7, B and C). The amplitude of ACh currents was significantly different in the presence of either Eps15 DN or Eps15 control, demonstrating the expression of the dominant-negative mutant (Fig. 7, C and E).

View larger version (24K): [in this window] [in a new window]   Fig. 7. Ivermectin increases surface expression of n7ACh receptors by a mechanism dependent on clathrin-dependent endocytosis. A, C, and D, ACh-evoked currents (300 µM) before and after 20-s application of 30 µM ivermectin (IVM) from oocytes expressing n7ACh receptors alone (A) and in combination either with control or DN Eps15 (C) or wild-type or DN AP-50 (D). B, histogram illustrating that the 2-fold potentiation of n7ACh currents by ivermectin is significantly decreased in the presence of the dominant-negative mutants of Eps15 or AP-50 proteins. E, means of the amplitude of ACh responses showing that expression of n7ACh receptors is higher in the presence of the dominant-negative mutants compared with the respective control proteins. F, surface expression of n7ACh-GFP receptors is increased by ivermectin. GFP-tagged n7ACh receptors expressing oocytes were incubated in IVM (30 µM) or in DMSO (c) 2 min before surface biotinylation. Immunoblot probes with anti-GFP antibodies shows that treatment by ivermectin increases the surface biotinylated fraction, whereas total fraction remains unchanged.

Likewise, coexpression of n7AChR and AP-50 DN also significantly reduced up-regulation of ACh currents by ivermectin. After preincubation with ivermectin, ACh currents represented 144.4 ± 8.35% (n = 12) of control currents (Fig. 7, B and D). Similar to P2X₄ receptors in oocytes coexpressing wild-type AP-50 and n7ACh receptors, ACh currents were rarely measurable, whereas in the presence of the dominant-negative AP-50 DN mutant, the amplitude of ACh currents was significantly higher (Fig. 7E). Thus, the functional up-regulation of n7ACh receptors by ivermectin did not occur when endocytosis was prevented.

To examine the surface expression of n7ACh receptors, we expressed GFP-tagged 7 receptors (7-GFP) in oocytes. We first verified that ivermectin potentiated 7-GFP receptor ACh current (not shown). Biotinylation and anti-GFP Western blot experiments showed that 2-min preincubation of oocytes expressing 7-GFP with 30 µM ivermectin increased the surface signal (by 240 ± 90%, two independent experiments; Fig. 7F). The surface fraction of 7-GFP subunits was estimated at 0.3% of total 7-GFP subunits in control conditions (incubation in 0.3% DMSO), revealing a weak surface expression. Overall, these experiments demonstrated that functional up-regulation of n7AChR by ivermectin can be accounted for by an increase in the number of surface receptors, which involves n7ACh receptor endocytosis.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Like many ligand-gated channels, including GABA_A and AMPA receptors (Carroll et al., 2001), P2X₄ receptors, but not other P2X subtypes tested so far, undergo rapid constitutive internalization by clathrin-dependent endocytosis and subsequent reinsertion into the plasma membrane (Bobanovic et al., 2002). The recruitment of P2X₄ channels to clathrin-coated pits is ensured by the direct association between a nontraditional tyrosine-based sorting signal in the cytosolic C-terminal tail of P2X₄ subunits and AP-50 (also called µ2) subunits of AP2 complexes (Royle et al., 2002, 2005). In this study, we show that disruption of the internalization signal of rat and human P2X₄ subunits induced a large increase in the amplitude of ATP-elicited current compared with P2X₄ WT currents by facilitating surface expression. According to previous studies on P2X trafficking in transfected neurons or HEK cells (Bobanovic et al., 2002), the disruption of the internalization motif results in an increase in the number of cell surface channels, indicating that a similar mechanism governs internalization of P2X₄ receptors in X. laevis oocytes.

Loss of Association with AP2 Produces Unique P2X₄ Functional Properties. Recombinant mammalian P2X₄ receptors are moderately desensitizing ATP-gated channels, insensitive to -methylene-ATP (Garcia-Guzman et al., 1997; Jones et al., 2000; North, 2002), and specifically potentiated by ivermectin (Khakh et al., 1999b; Priel and Silberberg, 2004). Internalization-deficient P2X₄ channels displayed a 15-fold increase in the apparent sensitivity to ATP (Fig. 2), a higher ionic permeability (Fig. 3), and in addition, these mutant receptors were not potentiated by ivermectin (Fig. 4). Previous studies demonstrated that high expression levels in transfected cells modify kinetics or agonist sensitivity of other ligand-gated channels such as P2X₂ or glycine receptors (Legendre et al., 2002; Fujiwara and Kubo, 2004). In this study, we took the precaution to reduce the expression of mutants to the level of wild-type P2X₄ receptors. Therefore, the modulation of P2X₄ channel properties, not attributable to channel density-dependent changes, is likely to be a direct consequence of the loss of interaction between AP2 complexes and the C terminus of P2X₄ receptors. Loss of interaction between microtubule-associated protein 1B and GABA_C receptors was also shown to increase the GABA sensitivity of GABA_C receptors (Billups et al., 2000). In our study, a higher responsiveness to -methylene-ATP was also observed but only at high channel density. Thus, contrary to other changes, the increase of -methylene-ATP potency may result from self-interaction or positive cooperation promoted by the aggregation of P2X₄ channels.

P2X₄ receptors change their ion selectivity: they open a small pore (I₁ state) in milliseconds and during prolonged exposure to agonists operate a transition toward a larger pore (I₂ state; Khakh et al., 1999a). In a low-Ca²⁺ solution, internalization-deficient P2X₄ channels displayed biphasic kinetics during prolonged ATP application, although I₂/I₁ ratio was significantly reduced. Determination of NMDG⁺/Na⁺ permeability changes revealed that I₁ state of P2X₄ Y378A channels was highly permeable to NMDG⁺ compared with wild-type P2X₄ channels. Fisher et al. (2004) demonstrated that the cytosolic domain of P2X₂ undergoes a conformational rearrangement correlating with permeability changes. It would be of interest to determine whether or not the loss of interaction between AP2 complex and P2X₄ channels promotes C terminus motion, leading to the opening in milliseconds of a highly permeable pore. It remains unclear why P2X₄ Y378A channels developed a (smaller) second current phase when no significant increase or decrease in NMDG⁺ permeability was noticed. Further investigations using P2X₄ Y378A receptors could be helpful to decipher mechanistic and dynamic aspects of the transition between both states.

Properties of Internalization-Deficient P2X₄ Channels Are Consistent with Those of Central ATP Responses. It is noteworthy that P2X₄ is the most widely distributed P2X subunit in the central nervous system (Norenberg and Illes, 2000; Bo et al., 2003). However, native P2X receptor phenotypes in the brain do not resemble heterologously expressed homo/heteromeric P2X₄ channels (North, 2002). For instance, in brainstem, habenula, or CA1 neurons, ATP- and -methylene-ATP-evoked currents are partially inhibited by suramin (Khakh et al., 1999b; Pankratov et al., 2002). Ivermectin does not modulate ATP-evoked current in brainstem or CA1 neurons (Khakh et al., 1999b), brain regions in which P2X₄ transcripts and/or proteins are abundant (Norenberg and Illes, 2000; Rubio and Soto, 2001). Kinetic and pharmacological properties of internalization-deficient P2X₄ channels described in this work are consistent with the phenotype of native P2X responses in many areas of the brain, suggesting that homomeric P2X₄ receptors may play an important role in fast ATP synaptic transmission. This implies that cellular processes (Traub, 2003) probably control interactions between the endocytic machinery and P2X₄ receptors and, consequently, the surface expression and function of synaptic P2X₄ receptors.

Mode of P2X₄ or n7ACh Receptors Potentiation by Ivermectin. Ivermectin increases ATP-evoked currents through rat and human P2X₄ channels and slows their deactivation in transfected HEK cells, oocytes, or neurons, but not through other P2X subtypes. In addition, in the presence of ivermectin, the ATP concentration-response curve is shifted to the left, and -methylene-ATP becomes a potent agonist of P2X₄ channels (Khakh et al., 1999b; Priel and Silberberg, 2004). Ivermectin has been described as a positive allosteric modulator with two potential binding sites on P2X₄ channels: a high-affinity site increases the maximum current, and a lower affinity site slows deactivation and increases apparent affinity for ATP (Priel and Silberberg, 2004). However, neither the binding sites nor the allosteric mechanism have been elucidated. In this study, we observed a 3-fold potentiation of maximum ATP current for rat and human P2X₄ WT channels, whereas P2X₄ Y378A or P2X₄ 377 channels of both species were not potentiated by ivermectin. However, ivermectin slows deactivation of both wild-type and mutated channels. Our data are consistent with the fact that changes in deactivation kinetics and up-regulation by ivermectin arise from two distinct mechanisms (Priel and Silberberg, 2004) and suggested a link between action of ivermectin and receptor internalization.

Biotinylation/immunoblotting experiments demonstrate that ivermectin treatment causes a 3- to 4-fold increase in the number of plasma membrane P2X₄ WT receptors without changing the total number of receptors. On the other hand, ivermectin does not change the surface expression of internalization-deficient receptors. Moreover, disruption of clathrin-dependent endocytosis by the expression of dominant-negative mutant of Eps15 or AP-50 protein prevented the potentiation of ATP current of P2X₄ WT receptors. Overall, these results demonstrated that the potentiation by ivermectin of P2X₄ current amplitude is attributable to receptors newly inserted into the plasma membrane by an endocytosis-dependent mechanism. Given that 1) ivermectin acts rapidly (2-min treatment), reversibly without changing the membrane capacitance (Khakh et al., 1999b), and 2) P2X₄ channels rapidly cycle in and out of the membrane (Bobanovic et al., 2002), it is tempting to speculate that ivermectin impairs P2X₄ receptor recruitment to coated pits via AP2 proteins (without preventing the formation of endocytic vesicles), which lead to the reinsertion into the plasma membrane of P2X₄ receptors from intracellular pools. Such an interpretation implies that during ivermectin treatment, surface P2X₄ receptors do not interact with AP2 complexes. This is in agreement with the finding that loss of interaction with AP2 complex conferred to P2X₄ receptors properties, which resemble those described in the presence of ivermectin (Khakh et al., 1999b). However, further studies will be necessary to bring direct evidence that the extracellular binding of ivermectin onto P2X₄ channels disrupts interaction with AP2 proteins. Although previous studies showed the constitutive endocytosis of P2X₄-GFP in HEK cells (Bobanovic et al., 2002), we noted that C-terminal-tagged P2X₄ receptors expressed in oocytes were less potentiated by ivermectin than wild-type P2X₄ receptors. A plausible explanation for these discrepancies is that fluorescent protein linked to the end of the short C terminus of P2X₄ subunits disturbs AP2/clathrin endocytosis in oocytes.

It remains to be determined whether an endogenous effector binds to ivermectin sites to regulate interaction between AP2 and P2X₄ channels. Such regulation may explain the ivermectin insensitivity of native P2X₄ channels (Khakh et al., 1999b) and the fact that ivermectin, widely used in human medicine against tropical filarial diseases, is well tolerated (Twum-Danso, 2003).

Ivermectin modulates other ligand-gated channels including GABA_A and n7ACh receptors (Krusek and Zemkova, 1994; Krause et al., 1998). Here we demonstrated that, similarly to P2X₄ receptors, ivermectin increases the number of surface n7ACh receptors by a mechanism dependent on clathrin/AP2-mediated endocytosis in X. laevis oocytes. These findings suggest that binding of ivermectin on the extracellular domain of distinct receptor-channels (such as GABA_A receptors, n7AChR) leads to the impairment of their constitutive internalization. Indeed, GABA_A receptors are recruited in clathrin-coated pits by interaction between dileucine or atypical signals and AP2 proteins (Herring et al., 2003). Despite recent findings showing a tyrosine kinasedependent regulation of the surface receptor density, regulation of surface n7ACh receptors and their internalization signals remains to be identified (Cho et al., 2005). Our results, showing the increase in n7ACh receptor expression in the presence of the dominant-negative mutant of Eps15 or AP-50, constitute the first indication that n7ACh receptors undergo constitutive internalization by a clathrin-dependent pathway.

	Acknowledgements

 
We thank Dr. Philippe Séguéla for providing rat n7AChR clones and Dr. Agnès Hémar for the dominant-negative mutants of Eps15cDNAs.

	Footnotes

 
This work was supported by Centre National de la Recherche Scientifique, Université Victor Segalen Bordeaux 2, Région Aquitaine, and by the Deutsche Forschungsgemeinschaft (So 390-1/2). E.T. is a recipient of a postgraduate fellowship from the Ministère de l'Education Nationale et de la Recherche.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.018812.

ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methyl-4-isoazoleproponic acid; AP, activator protein; n7AChr, nicotinic 7 acetylcholine; HEK, human embryonic kidney; ACh, acetylcholine; GFP, green fluorescent protein; NMDG, N-methyl-D-glucamine; DMSO, dimethyl sulfoxide; WT, wild type; DN, dominant negative; 7-GFP, expressed GFP-tagged 7.

¹ Current affiliation: Department of Pharmacological and Physiological Science, St. Louis University Medical School, St. Louis, MO.

Address correspondence to: Dr. Eric Boué-Grabot, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5543, Université Victor Segalen Bordeaux2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. E-mail: eric.boue-grabot{at}umr5543.u-bordeaux2.fr

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