Pless SA , Galpin JD , Niciforovic AP , Ahern CA .

56858 Canadian Institutes of Health Research , 56858-1 Canadian Institutes of Health Research

	Figure 2. Contributions of Asp 316 and Glu293(a) GV of natural (Asp, Asn) and unnatural (Nha) side-chains at position 316 (Asn316: V1/2 = 27.8 ± 3.3 mV; Nha316: V1/2 = −17.5 ± 2.3 mV; Asp316 (WT): V1/2 = −22.1 ± 0.7 mV, (n = 5–6)); insets show currents, electrostatic surface potential (ESP, red = −100 kcal/mol, green = 0 kcal/mol, blue = +100 kcal/mol) maps and energy minimized structures of side-chains; (b)/(c) Activation time constants (−80 mV to −25 mV) and deactivation time constants (+20 mV to −25 mV) for Asp316 and Nha316 (single exponential fit, n = 5); (d) GV of natural (Glu, Gln) and unnatural (Nha) side-chains at position 293; insets as in (a); (Gln293: V1/2 = −27.1 ± 0.5 mV; Nha293: V1/2 = −24.8 ± 0.8 mV; Glu293 (WT): V1/2 = −22.1 ± 0.7 mV (n = 4–6)); (e)/(f) Time constants of activation (e) and deactivation (f) for Glu293 and Nha293 (n = 5–7); protocol as in (b)/(c)); (g) Current (upper panel) and fluorescence (lower panel) recordings for Glu293 (WT) or Nha293 background, in black and green, respectively; (h) FV for Glu293 and Nha293 (V1/2: −63.1 ± 3.0 mV and 58.3 ± 1.8 mV, respectively; n = 4). Upper inset shows fluorescence sample traces (ON: −80 mV to −50 mV, OFF: +20 mV to −50 mV); lower inset shows averaged time constants (n = 4). Scale bars: 2 μA for current, 1 % for fluorescence and 50 ms for time (except 20 ms for Asn316). Asterisk indicates significant difference (p < 0.05).
	Figure 3. Glu293 and Asp316 do not contribute to a network of electrostatic charge-charge interactions(a) Model highlighting the proximity of Glu293 and Asp316 to Lys374 (PDB 2R9R); (b) Incorporating Nha simultaneously at positions 293 and 316 has only a minor effect on the GV (V1/2 = −27.2 ± 0.9 mV for Nha at positions 293 and 316 vs. V1/2 = −22.1 ± 0.7 mV for Glu in position 293 and Asp in position 316 (WT), n = 5); insets show currents, ESP maps and energy minimized structures with scale as in Fig. 2a; (c)/(d) Time constants of activation obtained from a depolarizing pulse from −80 mV to −25 mV (c) and time constants of deactivation obtained from a repolarizing pulse from +20 mV to −25 mV (d) for WT and for Nha simultaneously at positions 293 and 316 (all data fit with a single exponential, n = 5); asterisk indicates significant difference (p < 0.05); (e) Plot shows ab initio interaction energies at the HF/6-31G*+ level between Lys374 and Glu293 or Asp316 (open triangles or filled circles, respectively) in different dielectric environments. Side-chain moieties from PDB 2R9R were isolated, fixed and modeled as acetate (Glu and Asp) and methylammonium (Lys) in a variety of dielectric environments. All scale bars: 2 μA for current and 50 ms for time.
	Figure 4. Glu283 is likely to form a state-dependent electrostatic charge-charge interaction with S4 charges(a) Model highlighting the proximity of Glu283 to Arg 368 and Arg371 (PDB 2R9R); (b) Effects on GV by natural (Glu, Gln) and unnatural (Nha) side-chains at position 283 (V1/2 = 58.1 ± 2.0 mV for Gln283 (n = 4); V1/2 = 0.9 ± 2.1 mV for Nha283 (n = 8) and V1/2 = −22.1 ± 0.7 mV for Glu283 (WT)); insets show currents, ESP maps and energy minimized structures of natural and unnatural amino acids used at position 283 with scale for ESPs as in Fig. 2a; (c)/(d) Time constants of activation obtained from a depolarizing pulse from −80 mV to −25 mV (c) and time constants of deactivation obtained from a repolarizing pulse from from +20 mV (WT) or +40 mV (Nha283) to −25 mV (d) with Nha at positions 293 and 316 (all data fit with a single exponential, n = 5); asterisk indicates significant difference (p < 0.05); Scale bars: 2 μA for current, 50 ms for time.
	Figure 5. A cation-pi interaction in the potassium channel voltage-sensor(a) A model highlighting the proximity of Trp290 and Lys374 (PDB 3LNM); (b) Effect of fluorination at Trp290 on GV; insets show currents and ESPs for Trp (upper) and 4,5,6,7-F4-Trp (lower) (n = 5–7); scale for ESPs: red = −25 kcal/mol, green = 0 kcal/mol, blue = +25 kcal/mol; Scale bars: 2 μA for current, 50 ms for time; (c) Effect of fluorination at Trp290 on the Arg374 background; insets show currents and ESPs for Trp (upper) and 5,6,7-F4-Trp (lower) (n = 4–6); scale for ESPs as in (b); (d) Cation-pi plot for fluorination of Trp290 with Lys or Arg in position 374. ESPs for Trp derivatives are shown with scales as in (b).

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