McCracken ML , Gorini G , McCracken LM , Mayfield RD , Harris RA , Trudell JR .

R01 AA020980 NIAAA NIH HHS , R01 AA006399 NIAAA NIH HHS

	Figure 1. Effect of crosslinking on alcohol/anesthetic responses in TM1-3 mutants. Potentiation of EC5−10 glycine responses by 22 mM butanol (A) or 0.6 mM isoflurane (B) before and after application of the crosslinking agent HgCl2. HgCl2 (10 μM) was applied to wild-type, single mutants, and I229C/A288C and I229C/A288C/C290S mutants in the presence of 100 mM glycine (i.e., the activated/desensitized state). Values represent mean ± SEM from 4 to 5 oocytes. Repeated measures t-tests were used to test differences between pre-and post-HgCl2 conditions (*P < 0.05 compared to the pre-HgCl2 condition for each receptor).
	Figure 2. Effect of crosslinking on alcohol responses in TM3-4 mutants. Potentiation of EC5−10 glycine responses by 22 mM butanol before and after HgCl2. (A) HgCl2 (10 μM) was applied to wild-type, single mutants, and double mutants (A288C/W407C, A288C/Y410C) in the absence of glycine (i.e., the resting/closed state). (B) HgCl2 (10 μM) was applied to wild-type, single mutants, and double mutants (A288C/Y406C, A288C/I409C) in the presence of 100 mM glycine (activated/desensitized state). Values represent mean ± SEM from 4 to 5 oocytes. Data from wild-type and A288C receptors were combined across experiments and are represented in (A,B) and Figure 1A. Repeated measures t-tests were used to detect differences between the pre-and post-HgCl2 conditions (*P < 0.05 compared to the pre-HgCl2 condition for each receptor).
	Figure 3. Effect of crosslinking on alcohol responses in TM3-4 mutants. Tracings showing the effect of 22 mM butanol before and after application of 10 μM HgCl2 on EC5−10 glycine-induced currents in individual oocytes expressing wild-type (A) or A288C/Y410C (B) GlyRs. The upper bars indicate butanol pre-application.
	Figure 4. Effect of crosslinking on anesthetic responses in TM3-4 mutants. Potentiation of EC5−10 glycine responses by 0.6 mM isoflurane before and after HgCl2. (A) HgCl2 (10 μM) was applied to wild-type, double (A288C/W407C, A288C/Y410C), and single mutants in the absence of glycine (closed state). (B) HgCl2 (10 μM) was applied to wild-type, double (A288C/Y406C, A288C/I409C), and single mutants in the presence of 100 mM glycine (activated/desensitized state). Values represent mean ± SEM from 4 to 5 oocytes. Data from wild-type and A288C receptors were combined across experiments and are represented in (A,B) and Figure 1B. Repeated measures t-tests were used to detect differences between the pre-and post-HgCl2 conditions (*P < 0.05 compared to the pre-HgCl2 condition for each receptor).
	Figure 5. Effect of crosslinking using H2O2 on maximal glycine responses in TM1-3 mutants. The oxidizing agent, H2O2 (0.5%), was applied in the presence of 100 mM glycine for 1 minute followed by a 15-min washout. The response to glycine was measured and then the reducing agent dithiothreitol (DTT, 10 mM) was applied for 3 min followed by a 15-min washout. Values represent mean ± SEM from 3 to 6 oocytes (one-way ANOVA with Tukey's post-hoc test, *P < 0.05).
	Figure 6. Semiquantitative analysis of dimeric/monomeric band ratios resulting from uncrosslinked and crosslinked GlyRs. Crosslinking was obtained by applying 0.5% H2O2 by bath perfusion. Equal amounts of protein were extracted from oocytes, resolved by SDS-PAGE under non-reducing conditions, and transferred to a membrane where a GlyR alpha 1 antibody was used. Immunoblot images were processed using ImageJ64 software, and GLRA1-labeled band intensity was obtained for 50 and 100 kDa bands, which correspond to monomeric and dimeric GlyR subunits, respectively. Band intensities are reported as direct ratios (100:50 kDa). Immunoblot image analysis revealed a significant increase in the 100:50 kDa band ratio for the I229/A288C (TM1-3) double mutant after crosslinking, but not in the uncrosslinked mutant or in the corresponding wild-type samples. Conversely, no differences in the 100:50 kDa band ratios were detected between uncrosslinked (pre-H2O2) and crosslinked (post-H2O2) conditions for the A288C/Y410C (TM3-4) double mutant or in the corresponding wild-type samples. Statistically significant differences in band ratios between the pre-H2O2 and post-H2O2 conditions were measured and reported as a ratio (mean ± SEM; t-tests, *P < 0.05).
	Figure 7. GLRA1 homology models based on GluCl showing TM3-4 intra-subunit and TM1-3 inter-subunit distances. (A) The enlarged boxed area of a GLRA1 subunit shows distances between the C-alpha atoms of cysteines substituted at A288 (TM3) and Y406, W407, I409, and Y410 (TM4) within the same subunit were 14.7, 11.6, 15.3, and 14.6 Å, respectively. (B) The enlarged boxed area of two GLRA1 subunits shows distances between the C-alpha and S-S atoms of adjacent subunits (rendered as backbone ribbons in red and purple) were 10.3 and 7.8 Å, respectively.

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