Evidence indicating potential aromatic–aromatic interactions involving residue 248 of the pore helix and W216 of S5. (A) Inside-out current recordings demonstrating that the absence of aromatic–aromatic interactions resulting from the substitution of W216 by the nonaromatic Ala or Leu residue caused a strong increase in channel activity, with Pomax values of 0.68 ± 0.04 (n = 3) and 0.95 ± 0.01 (n = 3), respectively, compared with 0.22 ± 0.07 (n = 8) for the wild-type channel (W216). However, such behavior was not observed with the L215A mutant nor the T212A (not depicted). Substituting W216 by the smaller Phe aromatic residue (W216F) resulted in a single-channel fluctuation pattern characterized by a period of high activity (Pomax = 0.70 ± 0.2; n = 3), ending within <3 min in 50% of the recordings (n = 10) by a transition to stable closed state. Such behavior is compatible with strong aromatic–aromatic F248–W216F interactions maintaining the channel in the closed configuration. These results are summarized in the bar graph presented in C. These observations agree with a model whereby aromatic–aromatic interactions between F248 of the pore helix and W216 of S5 tend to stabilize the channel closed configuration, as suggested by the 3BEH-based model illustrated in B. 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. Illustration by Discovery Studio Visualizer (Accelrys).
