Fig. 1.

Subcellular distribution and functional properties of P2X constructs in HEK293 cells. A–F, The subcellular distribution of P2X4 WT (A), P2X4–GFP (C), P2X4(AU5) (E), P2X2 WT (B), P2X2-GFP (D), and P2X2(FLAG) (F) receptors in HEK293 cells. Cells expressing untagged receptors were stained using anti-P2X4/Cy3 or anti-P2X2/Cy3. Cells expressing P2X4(AU5) were stained using anti-AU5/Cy3 before (E, left panel) and anti-P2X4/FITC after permeabilization. Cells expressing P2X2(FLAG) were stained using anti-FLAG/Cy3 before (F, left panel) and anti-P2X2/FITC after permeabilization. Scale bar, 10 μm. G, Concentration–effect curves for ATP and representative traces showing ATP-evoked whole-cell currents for P2X4 (left) and P2X2 (right) constructs used in this study. ATP-evoked peak currents shown are normalized to compare the time course of desensitization. Calculated EC₅₀values were 3.8, 4.2, and 17 μm for P2X4 WT, P2X4–GFP, and P2X4(AU5), respectively. Calculated EC₅₀ values for P2X2 WT, P2X2–GFP, and P2X2(FLAG) were 3.9, 4.4, and 14.6 μm, respectively (n = 3–7 cells for each concentration).