Figure 3: Disulphide crosslinking between LF and DF impairs the activation of P2X4 receptors.

(a,b) Representative current recordings from cells expressing ^rV288C/T211C (a), and wild-type rP2X4 (b). Cells were voltage clamped at −60 mV, and currents were evoked by ATP (10 μM, 3 s) at 2 min intervals. Cells were perfused with dithiothreitol (DTT; 10 mM) and H₂O₂ (0.3%) at the times indicated. (c) Pooled data from the combination of experiments in a,b. y axis stands for the ratio of ATP-evoked currents after DTT treatments normalized by the currents before DTT treatment. Each line represents one paired measurements of an individual cell. Open boxes indicates mean±s.e.m., n=9–12; **P<0.01 after versus before DTT treatment, pair t-test. (d,e) Effects of disulphide crosslinking on the ATP-sensitivity of rP2X4^V288C/T211C. Representative current recordings (d) and pooled data (e) from cells expressing rP2X4^V288C/T211C show ATP-sensitivity of this double mutant before and after DTT treatments. Concentration–response relationship for ATP activation of rP2X4^V288C/T211C was obtained by measuring currents in response to different concentrations of ATP and all of the results used to generate a concentration–response relationship before and after DTT treatments were from the same group/cell by using amphotericin-perforated patch clamp. Data points are presented as mean±s.e.m. of 5–8 measurements, and the solid line are fit to Hill equation. The EC₅₀ and nH values of ATP for rP2X4^V288C/T211C in the absence and presence of DTT are 31.4±13.7 μM, nH=1.74±0.3, and 6.44±2.5 μM, nH=1.78±0.16, respectively.