In LeuT, deletion of V269 supports a HOIF conformation of the intracellular gate. Distance distributions of extracellular and intracellular spin-labeled Cys pairs reveal changes in the conformational equilibrium induced by ∆V269 in LeuT. (A) Extracellular reporter pairs (309–480) tagged on a 3D structure of LeuT. (D) Intracellular reporter pairs (7–86) tagged on a 3D structure of LeuT. (B and E) Distance distributions of the extracellular and intracellular reporter pair for LeuT in the apo conformation (black), in the presence of Na+ (red), or in the presence of Na+ plus Leu (blue). (C and F) Distance distributions of the extracellular and intracellular reporter pair for LeuT ∆V269 in the apo conformation (black), in the presence of Na+ (red), or in the presence of Na+ and Leu (blue).