Mutant cycle analyses demonstrating functional coupling between F248W of the pore helix and C277W or M273W of the S6 transmembrane segment. Energies are expressed in kilocalorie/mole. (A) Example of inside-out current recordings illustrating the effect of the M273W mutation on the change in Pomax resulting from the substitution at 248 of a Phe by a Trp. These observations are summarized in the cycle diagram illustrated in B, which shows that the mutation M273W resulted in a drastic Pomax increase (0.95 ± 0.005; n = 3), an effect that was partly impaired with a Trp at 248 (Pomax of 0.63 ± 0.05; n = 4). (C) Examples of inside-out current recordings illustrating the coupling between Trp at 248 and 277, respectively. The cycle diagram in D indicates in this regard that the mutation C277W totally prevented the Pomax increase normally resulting from the substitution F248W, while being ineffective in modifying Pomax with F248. These observations would be compatible with Trp–Trp interactions between F248W at the pore helix and C277W on S6 stabilizing the closed state (Fig. 11). On the basis of this analysis, it is concluded that both C277 and M273 are in proximity of F248. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. All-point histograms were computed from the entire recording.
