Figure 6.

P2X₂ receptor. Transient current obtained with membrane vesicles from a rP2X₂ HEK293 cell line after a 100 μM ATP concentration jump in the presence and absence of Na⁺ gradients. Black line: Inside directed gradient (Na⁺ outside, TMA⁺ inside vesicles). Blue line: outside directed Na⁺ gradient (TMA⁺ outside, Na⁺ inside vesicles). Red line: no gradient (TMA⁺ inside and outside vesicles). Experimental conditions: All solutions contained a basic buffer of 50 mM HEPES, 2 mM MgCl₂, pH 7.5 (TRIS). A 5-step solution exchange protocol was used consisting of the sequential application of solutions containing basic buffer plus compounds as given below. For the inside directed Na⁺ gradient the protocol was (duration given in parentheses): 100 mM TMACl(1 s) − 100 mM NaCl(5 s) − 100 mM NaCl + 100 μM ATP(2 s) (this phase is shown in the figure) − 100 mM NaCl(2 s) − 100 mM TMACl(5 s). To ensure optimal regeneration, the sensors were flushed again for 1 s with 100 mM TMACl 30 s after each measurement and a delay time of 60 s between the measurements was kept. For the outside directed Na⁺ gradient TMACl and NaCl were interchanged. In the absence of a Na⁺ gradient all solutions contained TMACl. Inset: Western blot of rP2X₂ HEK293 cells. Lane 1: Cell lysate after 24 h tetracycline induction. Lane 2: Lysate of noninduced cells. Lane 3: Molecular weight marker.