Figure 2. Arg 555 and Thr 1246 become energetically coupled upon spontaneous pore opening.
(a–d), Microscopic currents at −80 mV, and their dependence on ATP and PKA (black bars), for (a) the P355A-K978C background construct, and for this background construct carrying (b) the R555K or (c) T1246N single mutations or (d) the R555K-T1246N double mutation. (e–g), Spontaneous open probabilities (e, n = 19–21), opening rates (f, n = 15–19), and closing rates (g, n = 15–19) of the background construct (black bars), and of constructs carrying additional R555K (blue bars), T1246N (red bars), or R555K-T1246N (violet bars) mutations. (h–j), Thermodynamic mutant cycles showing mutation-induced changes (h) in the stability of the spontaneous open state relative to the closed state, and (i–j) in the height of the activation free energy barriers for spontaneous opening (i) and closure (j). Each corner is represented by the side chains at positions 555 and 1246; k, Boltzmann's constant, T, absolute temperature.