Macdonald LC , Kim RY , Kurata HT , Fedida D .

	Figure 1. Aromatic residues Tyr652 and Phe656 in hERG shown to be important in drug block. (A) Cartoon schematic of approximate location of Tyr652 and Phe656 in hERG pore region. (B) Top-down view of the pore region of hERG pore from cryo-EM structure 5VA132. Aromatic residues Tyr652 and Phe656 are highlighted in blue and pink respectively. (C) Chemical structures of terfenadine, quinidine, and dofetilide.
	Figure 2. Incorporation of natural and unnatural amino acids through nonsense suppression is well tolerated at both Tyr652 and Phe656. A 5 s depolarization to 0 mV from a holding potential of −110 mV was used to assess construct expression. All mutants were successfully expressed via nonsense suppression and representative traces are displayed here.
	Figure 3. Voltage-dependence of activation is unchanged by Phe fluorination at position 652, but hyperpolarized at position 656. (A) Ionic current from WT, Y652F2, and F656F2 mutant hERG channels recorded during 5 s depolarizations from −110 mV to 20 mV in 10 mV increments followed by repolarization to −110 mV (protocol inset at top). Pulses were applied every 7 s. (B) GV0.5 values for all constructs. GV fit parameters are found in Table S1. (C) GV relationships of the UAA mutants at position 656 and WT as measured from tail currents at −110 mV.
	Figure 4. Voltage-dependence of inactivation unchanged among mutant channels. (A) Representative trace of WT hERG current resulting from the inactivation protocol (inset left). The protocol is described in further detail in Methods. An expanded view of the currents during the P2 and P3 pulses are shown in the right inset. (B) V0.5 values for inactivation of all constructs. Fit parameters are found in Table S1. (C) Steady-state inactivation relationships of all constructs.
	Figure 5. Fluorination of Phe656 does not reduce terfenadine potency; however, mono-fluorination of Y652F at the C4 phenyl ring carbon results in a large reduction in terfenadine potency. (A) Protocol used to evaluate drug block. From a holding potential of −110 mV, channels are depolarized to 0 mV for 5 s and are then repolarized first to −50 mV for 300 ms and then back to −110 mV. (B) Currents from Y652F*, F656F*, and Y652F1 in control and in response to terfenadine concentrations of 10, 30, 100, 300, 1000 nM. (C) Terfenadine IC50s and Hill coefficients. IC50 and h fit parameters are found in Table S2. (D) Concentration-response curves of WT and Y652F series.
	Figure 6. Fluorination of Phe656 reduces quinidine potency in a non-additive fashion; however, only mono-fluorination of Y652F at the C4 phenyl ring carbon results in reduced quinidine potency. (A) Currents from Y652F2 and Y652F1 elicited from the protocol shown in Fig. 4A in control and in response to quinidine concentrations of 2, 6, 20, 60, 200 μM (B) Quinidine IC50 and Hill coefficients. IC50 and h fit parameters are found in Table S2. (C) Concentration-response curves of Y652F fluorinated mutant series and WT (D) Concentration-response curves of Phe656 fluorinated mutant series and WT.
	Figure 7. Fluorination of Phe656 does not reduce dofetilide potency; however, mono-fluorination of Y652F at the C4 carbon of the phenyl ring results in a large reduction in dofetilide potency. (A) Currents from Y652F1 and F656F1 in control and in response to dofetilide concentrations of 10, 30, 100, 300, 1000 nM. (B) Dofetilide IC50 and Hill coefficients. IC50 and h fit parameters are found in Table S2. (C) Concentration-response curves of WT and Y652F series.

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