Schneider M et al. (2017), Interaction of Purinergic P2X4 and P2X7 Recepto...

XB-ART-54338

Front Pharmacol 2017 May 26;8:860. doi: 10.3389/fphar.2017.00860.

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Interaction of Purinergic P2X4 and P2X7 Receptor Subunits.

Schneider M , Prudic K , Pippel A , Klapperstück M , Braam U , Müller CE , Schmalzing G , Markwardt F .

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P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1-P2X7) that form homo- and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X41-384 and C-terminally truncated fluorescent P2X71-408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration-response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.

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Species referenced: Xenopus laevis
Genes referenced: p2rx2 p2rx4 p2rx7 rel slco1a2

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	FIGURE 1. Fluorescence signals before and after acceptor photobleaching. Example of Donor (GFP) recovery after acceptor (RFP) photobleaching for the P2X4 construct with a C-terminal tandem GFP-RFP label. (A,B) Pre-bleaching (A) and post-bleaching (B) fluorescence images were obtained as described in the Methods section using a confocal laser scanning microscope. The slightly increasing GFP fluorescence is shown on the left side and the RFP fluorescence, which is strongly decreased due to application of the maximal RFP exciting light for 1 min on the right side. (C) The FRET signal was calculated pointwise using the microscope software in the same manner as shown in Figure 2. A strong FRET signal is measured in areas of RFP bleaching. Blue pixels indicate low and yellow pixels high FE.
	FIGURE 2. Förster resonance energy transfer (FRET) between GFP- and RFP -labeled P2XR subunits measured with donor recovery by acceptor photobleaching. Measurements were made at the oocyte pole adjacent to the bottom of the recording chamber (P) or at the equator (E) of the oocytes, as shown in Figure 1. The degree of donor recovery was plotted against the degree of acceptor bleaching. FRET efficiency (FE) was obtained by extrapolating the linear regression line to complete acceptor bleaching (right hand y-axis). (A) Negative control: coexpression of C-terminally RFP-labeled hP2X4 with SP-GFP-GLYRA1. (B) In oocytes expressing either hP2X4-GFP or hP2X4-RFP, the mean changes in the donor (donor recovery) and acceptor fluorescence (acceptor bleach) were measured at bleached versus non-bleached areas. The decrease of the donor fluorescence in regions of acceptor bleach indicates a bleaching effect of the acceptor excitation light and leads to calculation of negative FE values. (C) Positive control: P2X4 protein with a C-terminal tandem GFP-RFP label. (D) Coexpression of C-terminally GFP- and RFP-labeled hP2X4 constructs. Mean fluorescence values were calculated from 50–60 regions of interests (ROIs) in 5–10 oocytes.
	FIGURE 3. Förster resonance energy transfer measurement of P2X4 and P2X7 subunit interactions. FRET efficiencies were calculated as described in Figure 2. Horizontal line represents GFP bleaching during acceptor bleaching leading to negative FE values of –0.019 (see Figure 2B). ∗, FRET efficiencies significantly larger than –0.019. Data are the means ± SEM from 12–20 oocytes. X and Y denote short, flexible linkers (-GAGA- and -AGAG-, respectively; single amino acid letter code) flanking the GFP or RFP moiety. The position of the fluorophore name indicates the position of the label in the P2X receptor fusion construct (i.e., P2X7-GFP indicates a C-terminal label). Bar colors: green = negative control, blue = P2X4 constructs, red = P2X7 constructs, pink = P2X4/P2X7 coexpression. Bar patterns: cross-hatched = tandem GFP-RFP label, checkered = positive controls.
	FIGURE 4. Functional testing of fluorescence-labeled P2X4 and P2X7 constructs by two-electrode voltage clamp measurements. (A) Examples of current traces in oocytes expressing wildtype P2X4 (P2X4wt) of P2X7 (P2X7wt) or different fluorescence-labeled P2X4 and P2X7 constructs, as indicated. The colors of the construct names relate to the colors of the bars in (B,C). (B) Maximal current amplitudes obtained during the application of 1 mM ATP for 6 s. (C) Kinetics of ion channel currents in oocytes expressing different labeled P2X constructs. The relative inactivation (Iinact,rel) was calculated as the amplitude of the current between 2 and 6 s after the application of 1 mM ATP normalized to the current amplitude at the 2 s time point. A decay in the current amplitude (Iinact,rel > 0) indicates desensitization (seen for all P2X4 constructs); an increase in current amplitude during this period (Iinact,rel < 0) was usually seen for P2X7 constructs and is of unknown origin. Bar colors: light blue = P2X4wt or C-terminally labeled constructs, dark blue = P2X4 constructs labeled within the extracelllar domain, red = P2X7wt or C-terminally labeled constructs, rosy = C-terminally truncated and labeled P2X7 constructs, dark red = P2X7 constructs labeled within the extracelllar domain. Data are the means ± SEM from 5–20 oocytes.
	FIGURE 5. Kinetics of ion channel currents in hP2X4/hP2X7-coexpressing oocytes. Two-electrode voltage clamp measurements of currents in Xenopus oocytes expressing hP2X4 and hP2X7 receptors were elicited by the application of 0.1 mM ATP. (A) Mean current traces were recorded from oocytes expressing hP2X4 or hP2X7 subunits alone or together, as indicated. (B) Mean currents in oocytes coexpressing hP2X4 and hP2X7 were compared with the sum of those in oocytes expressing hP2X4 or hP2X7 alone. The mean current amplitudes were not significantly different at any time point, indicating that P2X4/P2X7 heteromers with distinct current kinetics are not formed. Data are the means ± SD from five oocytes at each time point.
	FIGURE 6. Dependence on the ATP concentration of coexpressed hP2X4 and hP2X7 subunits. (A) Maximal current amplitudes in the presence of various ATP concentrations ([ATP]) were obtained by two-electrode voltage clamp measurements in oocytes expressing hP2X4 and/or hP2X7, as indicated. These amplitudes were measured during a 12 s lasting ATP application and normalized to the amplitude during a preceding application of 0.1 mM ATP to yield the relative current amplitude Irel. Irel values for hP2X4- or hP2X7-expressing oocytes are significantly different at all ATP concentrations. Current amplitudes of oocytes expressing hP2X4 + hP2X7 are significantly different from those of P2X7-expressing oocytes at 0.1–1 μM ATP and significantly different from those of P2X4-expressing oocytes at 0.01–1 mM ATP. Thus, the ATP concentration–response curve for hP2X4/hP2X7-coexpressing oocytes is dominated by the hP2X4 component at low ATP concentrations and by the P2X7 component at high ATP concentrations, arguing against a distinct P2X4/P2X7 phenotype regarding the agonist sensitivity. (B–D) Averaged current traces of different oocytes expressing hP2X4 and hP2X7, elicited by the second ATP application at the indicated concentrations. Data are the means ± SD from 4–10 oocytes.
	FIGURE 7. Effect of ivermectin on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. (A–C) Two-electrode voltage clamp measurements showing the effect of ivermectin (3 μM) on ATP-induced currents of oocytes expressing (A) hP2X4, (B) hP2X7, or (C) hP2X4 + hP2X7. Oocytes were incubated for 1 min with 3 μM ivermectin, and then with 3 μM ivermectin + 0.1 mM ATP. (D) Current amplitudes measured in P2X4/P2X7-coexpressing oocytes during ivermectin application were compared with the sum of the P2X4- and P2X7-dependent current amplitudes (as shown in parts A,B, respectively). Mean current amplitudes were not significantly different at any time point, indicating the lack of a distinct P2X4/P2X7 phenotype related to ivermectin. This may be explained by ivermectin affecting only homomeric P2X4 receptors. Data are the means ± SD from 4–6 oocytes.
	FIGURE 8. Effect of PSB-14517 on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. Two-electrode voltage clamp measurements showing ATP-induced current amplitudes (Iact,3s) at 3 s after the application of 0.1 mM ATP. The theoretical current remaining after PSB-14517 application to hP2X4/hP2X7-expressing oocytes (rightmost column) was calculated as described in Section “Results.” The theoretical current amplitude was not significantly different from the measured value (next to rightmost column), indicating the lack of a distinct P2X4/P2X7 phenotype related to PSB-14517, which may be explained by PSB-14517 affecting only homomeric P2X4 receptors. Data are means ± SD from 4–11 oocytes.
	FIGURE 9. Effect of BzATP on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. (A–C) Two-electrode voltage clamp measurements showing the effect of BzATP on ATP-induced currents of oocytes expressing (A) hP2X4, (B) hP2X7, or (C) hP2X4 + hP2X7. (D) BzATP-induced currents measured in P2X4/P2X7-coexpressing oocytes were compared with the sum of P2X4- and P2X7-dependent current amplitudes (shown in parts A,B, respectively). Mean current amplitudes were not significantly different at any time point, indicating the lack of a distinct P2X4/P2X7 phenotype related to BzATP stimulation. means ± SD from 5–6 oocytes.
	FIGURE 10. Effect of Mg2+ on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. (A–C) Two-electrode voltage clamp measurements showing the effect of Mg2+ on ATP-induced currents of oocytes expressing (A) hP2X4, (B) hP2X7, or (C) hP2X4 + hP2X7. (D) Currents induced by the coapplication of ATP and 1.5 mM Mg2+ measured in P2X4/P2X7-coexpressing oocytes were compared with the sum of P2X4- and P2X7-dependent current amplitudes (shown in parts A,B, respectively). Mean current amplitudes were not significantly different at any time point, indicating the lack of a distinct P2X4/P2X7 phenotype related to Mg2+ inhibition. Data are means ± SD from 4–6 oocytes.
	FIGURE 11. Effect of oATP on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. (A–C) Two-electrode voltage clamp measurements showing the effect of 3 h preincubation in 0.3 mM oATP on ATP-induced currents of oocytes expressing (A) hP2X4, (B) hP2X7, or (C) hP2X4 + hP2X7. (D) Currents induced by ATP after oATP preincubation in P2X4/P2X7-coexpressing oocytes were compared with the sum of P2X4- and P2X7-dependent current amplitudes (shown in parts A,B, respectively). Mean current amplitudes were not significantly different at any time point, indicating the lack of a distinct P2X4/P2X7 phenotype related to oATP antagonism. Data are the means ± SD from 4–5 oocytes.
	FIGURE 12. Effect of the P2X7 antagonist A438079 on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes. Two-electrode voltage clamp measurements of ATP-induced current amplitudes (Iact,3s) at 3 s after the application of 0.1 mM ATP. Current amplitudes of the first and second ATP application are denoted (1) and (2), respectively. The theoretical current remaining after applying A438079 to P2X4/P2X7-expressing oocytes (rightmost column) was calculated as described in Section “Results.” The theoretical current amplitude was not significantly different from the measured value (next to rightmost column), indicating the lack of a P2X4/P2X7 phenotype related to A438079, which may be explained by A438079 affecting only homomeric P2X7 receptors. Data are means ± SD from 4–11 oocytes.

References [+] :

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