After removal of posterior hyaloid, 0.2 mL Brilliant Blue G was applied on the macula, to stain epiretinal membraneunder air conditions for 2 minutes.

Whole-cell recordings were made 20 to 48 h after transient transfection and 24 to 72 h after passage of stable cells, using an EPC9 patch clamp amplifier (HEKA Elektronik, Lambrechet, Germany). Unless otherwise noted, membrane potential was held at 260 mV. Recording pipettes (4–7 MV) were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL) and filled with an intracellular solution that consisted of (in mM): 145 NaF, 10 EGTA, 10 HEPES. The external solution contained (in mM): 147 NaCl, 10 HEPES, 13 glucose, 2 KCl, 2 CaCl2, and 1 MgCl2. Osmolarity and pH values of both solutions were 300 to 315 mOsm/l and 7.3, respectively. All the experiments presented here were performed at room temperature. Agonists and Brilliant Blue G were applied using an RSC 200 fast-flow delivery system (Biologic Science Instruments, Grenoble, France). Agonists were applied every 2 min except for experiments on P2X1, P2X3, and rat P2X4, in which 3- to 8-min intervals were used because of the prolonged rundown of these responses. The duration of agonist application was 1 s for the P2X1 receptor, 2 s for the P2X2, P2X3, and P2X2/3, and P2X4 receptors, 3 s for the P2X1/5 receptor, and 4 s for the P2X7 receptor. For all except the P2X1 receptor, repetitive stimuli at the frequencies noted above were applied in the absence of Brilliant Blue G until evoked currents were stable (65%); the clamped cell was perfused with Brilliant Blue G (0.1–10 mM) for 4 min, and current evoked by agonist was measured. The peak current amplitude was expressed as a percentage of the amplitude obtained under the control conditions. Because of the marked rundown of currents at the P2X1 receptor, Brilliant Blue G was added to the cells for 4 min after the second application of agonist. The ratios of the peak amplitude induced by the third application of agonist relative to the peak amplitude by the second application of agonist were calculated and compared in the absence and presence of Brilliant Blue G.