Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

Abstract

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of approximately 430, approximately 580 and approximately 760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of approximately 430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.

Fig. 1

Supramolecular organization of P2X4R, P2X7R and Cav-1. Native membrane extracts from E10 (a–c), L2 (c) and A549 cells (c) were isolated and solubilized with digitonin. Solubilization of native membranes was identical for hrCN-PAGE and BN-PAGE. Proteins were separated by 2D-BN/SDS-PAGE (a) or hrCN-PAGE (b, c), blotted and probed with antibodies against P2X7R, P2X4R and Cav-1. Sizes of the molecular mass standards are indicated

Fig. 2

Coimmunoprecipitation of P2X7R and Cav-1 and P2X7R and P2X4R. Native membrane protein extracts were solubilized with 1% NP-40 (a) or 1% digitonin (b) and immunoprecipitated with anti-P2X7R (a, b, left side) or anti-P2X4R antibody (a, b, right side) and analyzed by Western blotting with anti-P2X7R, anti-P2X4R and anti-Cav-1 antibodies. For comparison, immunoprecipitate (IP; lane 1) and supernatant of the immunoprecipitate (S; lane 2) were loaded. Lane 3 shows the immunoprecipitate of the negative control and its supernatant was loaded in lane 4. In addition, the PVDF membrane was probed with anti-P2X7R or anti-P2X4R antibodies to ensure appropriate binding of P2X7R or P2X4R protein to the antibody-Dynabead complex. A representative result from three independent experiments is shown

Fig. 3

The effect of P2rx7 downregulation in E10 cells. After transfection of E10 cells with three different shRNA constructs targeting P2rx7 (shRNA1-3, a) and three different scrambled shRNAs (scshRNA1-3, b), cells were harvested and P2X7R, P2X4R and Cav-1 protein levels were analyzed by Western blot analysis with rabbit polyclonal anti-P2X7R antibody, rabbit polyclonal anti-P2X4R antibody or mouse monoclonal anti-Cav-1 antibody. γ-Tubulin served as a loading control. Representative data from three separate experiments are shown. Statistical analysis of P2X7R, P2X4R and Cav-1 protein levels are shown on the right side. *p < 0.05; **p < 0.01

Fig. 4

Distribution and localization of P2X4R and Cav-1 after P2rx7 downregulation. a Western blot analysis (using an anti-P2X4R antibody (left side) or an anti-T1α antibody (right side) of biotinylated whole cell lysates (total) and plasma membranes (surface) obtained from untreated cells (control) and after P2rx7 downregulation in E10 cells. A representative Western blot (n = 3) is depicted. The standard protein GAPDH was used to confirm equal protein loading. b Immunofluorescence demonstration of endogenous Cav-1 (Cy3) in untransfected E10 cells (left side) and in E10 cells transfected with P2rx7 shRNA3 (right side). Scale bar corresponds to 20 μm. c Native membrane extracts from untransfected E10 cells and E10 cells transfected with P2rx7 shRNA3 were isolated and solubilized with digitonin. Proteins were separated by hrCN-PAGE, blotted and probed with antibodies against P2X7R, P2X4R and Cav-1

Fig. 5

The effect of P2rx4 downregulation in E10 cells. After transfection of E10 cells with three different shRNA constructs targeting P2rx4 (shRNA1-3, a) and three different scrambled shRNAs (scshRNA1-3, b), cells were harvested and P2X4R, P2X7R and Cav-1 levels and were analyzed by Western blot analysis with rabbit polyclonal anti-P2X4R antibody, rabbit polyclonal anti-P2X7R antibody or mouse monoclonal anti-Cav-1 antibody. γ-tubulin served as a loading control. Representative data from three separate experiments are shown. Statistical analysis of P2X4R, P2X7R and Cav-1 protein levels are shown on the right side. *p < 0.05; **p < 0.01

Fig. 6

Distribution and localization of P2X7R and Cav-1 after P2rx4 downregulation. a Western blot analysis (using an anti-P2X4R antibody (left side) or an anti-T1α antibody (right side) of biotinylated whole-cell lysates (total) and plasma membranes (surface) obtained from untreated cells (control) and after P2rx4 downregulation in E10 cells. A representative Western blot (n = 3) is depicted. The standard protein GAPDH was used to confirm equal protein loading. b Immunofluorescence demonstration of endogenous Cav-1 (Cy3) in untransfected E10 cells (left side) and in E10 cells transfected with P2rx4 shRNA3 (right side). Scale bar corresponds to 20 μm. c Native membrane extracts from untransfected E10 cells and E10 cells transfected with P2rx4 shRNA3 were isolated and solubilized with digitonin. Proteins were separated by hrCN-PAGE, blotted and probed with antibodies against P2X7R, P2X4R and Cav-1

Fig. 7

Membrane lipid-binding assay. From left to right: lipid-coated membrane strips were incubated with GST-P2X4R C-terminal fusion protein (GST-P2X4R-CT) and GST-P2X7R C-terminal fusion protein (GST-P2X7R-CT), GST alone and N-terminal GST-tagged recombinant PLC-δ1 PH domain (GST-PI(4,5)P₂ Grip™). GST showed no specific binding on immobilized lipids, but the C-terminus of P2X4R and P2X7R bound directly to PA, PS, PG, 3-sulfogalactosylceramide, cardiolipin and the phosphoinositides PI(4)P, PI(4,5)P₂ and PI(3,4,5)P₃. N-terminal PLC-δ1 PH domain was used as positive control