Structure representations of single and double mutants supporting close proximity between C277 and M273 of S6 and F248 of the pore helix. Representation of the channel in the closed state (3BEH template). (A) The relative orientation of the Thr (W) for the F248W–C277W mutant suggests possible π–π interactions that would stabilize the pore helix in the closed configuration, thus accounting for the mutation C277W preventing the Pomax increase coming from the F248W mutation. (B) Also illustrated is the tight packing coming from the mutation M273W, which confirms the proximity of F248 and M273, as demonstrated through the double mutant cycle analysis in Fig. 10. Illustrations by Discovery Studio Visualizer (Accelrys).