Removing the negative ESP of the S2 aromatic has minimal functional consequences. (A) Chemical structures and ESP maps of Phe and 3,4,5-trifluoro-Phe (F₃-Phe) (ESP: red, −25 kcal/mol; green, 0 kcal/mol; blue, +25 kcal/mol; see Pless et al., 2011a, for details). (B) Sample traces for currents recorded from WT or mutants in which F₃-Phe has been introduced in the S2 HC (DI, Tyr168TAG + F₃-Phe; DII, Phe621TAG + F₃-Phe; DIII, Phe1076TAG + F₃-Phe; DIV, Phe1396TAG + F₃-Phe). Bars: horizontal, 5 ms; vertical, 500 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. (C and D) G-V (C) and SSI curves (D) for WT and mutants in which the S2 aromatic was replaced by F₃-Phe; the inset in D shows a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the F₃-Phe mutants in DI–DIV.