Fig. 2: Binding of adenine nucleotides in ClC-3 exchanger.
a–d The whole-cell current recordings of ClC-3 after about five minutes of break-in with 10 mM ATP, ATPγS, ADP, or AMP in pipette solution. The black represents the initial, the colored represents the current after five minutes. The voltage ramp protocol (from −120 to +180 mV in 450 ms) was used. e The current ratio stimulated by 10 mM ATP, ATPγS, ADP, and AMP to the initial current (mean ± SEM, n = 5 from biologically independent cells). Two-sided t-test, the exact P values were labeled. f–h The structure of mClC-3ATP. The electron density of ATP are shown in red. i The ATP binding pocket of ClC-3. j The current ratio after about five minutes of 10 mM ATP stimulation to the initial current of ClC-3 mutants (based on the mClC-3bS3/S2/S1 background).The corresponding n values are 5, 3, 5, 5, 5, 3, 6, 3, 3, 3, 3, 4, 5, 4, 4, 3, 5, 4, 3, respectively (mean ± S.E.M. from biologically independent cells). Two-sided t-test, the exact P values were labeled. k The backbone of the ATP binding site comparing the apo (brown) with the ATP (green) bound models. l The structure of mClC-3ADP. The electron density of ADP are shown in blue. m The structure of mClC-3AMP. The electron density of AMP are shown in orange. n The overlay of the ATP (red), ADP (blue) and AMP (orange) bound models. H89 and K798 close to gamma phosphate in ATP are shown. o The backbone of the ATP binding site comparing the ATP (green), ADP (grey), and AMP (pink) bound models. p A concentration-response for AMP or AMP-PNP with ATP showing that these nucleotides antagonise the current induced by ATP in a concentration-dependent manner. The ratio of the current after about five minutes of different concentration of AMP or AMP-PNP with 10 mM ATP to the initial. The corresponding n values are 5, 5, 6, 5, 5, 5, 5, 5, 5, respectively (mean ± S.E.M. from biologically independent cells). Two-sided t-test, the exact P values were labeled.
