Permeability of recombinant P2X2Rs. Rat P2X2aR, P2X2bR, and P2X4R were expressed in HEK293 (A and B) or GT1 (C) cells, and recording was done in single cells 24 h after transfection using whole cell patch-clamp mode (A and B) or imaging techniques (C), as described in Materials and methods. All experiments were done with naive cells, i.e., during initial agonist application. (A) Patterns of ATP (100 µM)-induced current in HEK293 cells bathed in medium containing 155 mM NMDG⁺, 10 mM HEPES, and 10 mM glucose only. Currents were recorded at a −60-mV holding potential during 60-s agonist application. Note the lack of inward current in agonist-stimulated P2X4Rs. (Inset) Temporal correlation between P2X2aR current (black) and reversal potential (gray). (B) Positive shifts in reversal potential observed during the initial 25-s agonist application in HEK293 cells bathed in the same medium. In experiments with P2X2aR- and P2X4R-expressing cells, 0.485-s voltage ramps were delivered twice per second from a holding potential of 0 mV (P2X2aR) or −60 mV (P2X4R); 10 out of 50 traces for the I-V relationship with equal time intervals are shown (progressing from left to right). In experiments with P2X2bR, 48.5-ms voltage ramps were delivered twice per 100 ms from a holding potential of 0 mV; the first 15 traces for the I-V relationship with equal time intervals are shown. (C) Effects of ATP on Fura-2 leak in GT1 cells bathed in Ca²⁺-deficient KR buffer. Normalized fluorescence intensities (gray, λ_ex = 380; black, λ_ex = 340 nm) are shown.