Rienzo M , Rocchi AR , Threatt SD , Dougherty DA , Lummis SC .

T32 GM007616 NIGMS NIH HHS , NS 34407 NINDS NIH HHS , R01 NS034407 NINDS NIH HHS , WT 81925 Wellcome Trust , R37 NS034407 NINDS NIH HHS , GM07616 NIGMS NIH HHS

	FIGURE 1. Sequence alignment of GLIC, ELIC, and two example eukaryotic pLGIC subunits: nAChR α7 and GABAA R α1. Prolines examined in this study are shown in red. Shaded residues are conserved or similar in side-chain character among sequences. The M3/M4 loop of the eukaryotic receptors has been removed at *.
	FIGURE 2. Proline sites examined in this study. A, GLIC subunit structure, indicating proline residues (PDB: 3EHZ). B, conventional mutagenesis of GLIC Pro residues. Substitution with Ala mostly had little or no effect on pH50 values, although P119A and P203A were nonfunctional, and P198A had increased sensitivity. P6-9A indicates simultaneous substitution of prolines at sites 6, 7, 8, and 9 with Ala; individual Ala substitutions at these sites yielded similar pH50 values (not shown). Data shown are mean ± S.E., n = 3–4. *, pH-induced responses were comparable with uninjected cells. Typical maximal responses for WT and all Ala-containing mutants were 20–40 μA, except for P14A, where maximal responses were 2–5 μA.
	FIGURE 3. The GLIC YPF motif. A, the ECD/TM interface in GLIC, highlighting the YPF motif in the cis conformation (PDB: 3EAM). B, relationship between mean pH50 values and cis-trans preferences for Pro analogs at Pro-119 in GLIC (filled circles), when compared with the analogous position (Pro-136) in the muscle-type nAChR (open squares) (17). Typical maximal currents for functional mutants generated by nonsense suppression were 0.5–2 μA, with P119Pip giving responses as high as 10 μA.
	FIGURE 4. Structures of amino acid analogs used in this study.
	FIGURE 5. The GLIC M1 prolines. A and B, Pro-198 and Pro-203 in the top of the M1 helix at pH 7 (PDB: 4NPQ) (A) and pH 4 (PDB: 3EHZ) (B). Shown are a hydrogen bond between Asn-199 and the main-chain carbonyl of Ser-195 (black), the disrupted hydrogen bond hypothesized to be restored by conventional mutagenesis at Pro-198 (red), and the interhelix hydrogen bond between His-234 and the Ile-258 carbonyl (green). C and D, pH50 values for GLIC Pro-198 (C) and Pro-203 (D) mutants. Data shown are mean ± S.E., with n = 8–20. Conventional mutants at Pro-198 gave currents comparable with wild type GLIC. Typical maximal currents from functional mutants generated by nonsense suppression were 0.5–3 μA, with P198Lah giving responses as high as 20 μA. *, pH-induced responses comparable with uninjected cells; §, a biphasic curve was observed with a low-sensitivity component having pH50 < 4.5; ‡, currents were observed, but pH50 was too low to measure (< 4.5) due to nonspecific acid-induced currents.
	FIGURE 6. GLIC Pro-299. A, The GLIC TM domain, highlighting Pro-299 (PDB: 3EHZ). B, pH50 values for GLIC Pro-299 mutants. Data shown are mean ± S.E., with n = 7–20. Typical maximal currents from functional mutants generated by nonsense suppression were 0.5–5 μA. *, pH-induced responses comparable with uninjected cells.

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