Figure 4.

Molecular Dynamic Simulation, Mutagenesis, and Correlation Analysis

(A) Overview of residues forming H-bond between GB chain and GR chain in the GB/GR and GB/GRM complexes at the end of the simulation. GB chain and residue labels are colored in cyan. GR chain and residue labels are colored in pink. H-bonds are shown by the red dashed line.

(B) Binding energy (kcal/mol) between subunits in various composing form of dimers.

(C) Number of H-bonds formed between GB chain and GR chain in the GB/GR and GB/GRM complexes. The data are shown as averages of each 200 ps. Data are represented as mean ± SD.

(D) VDW contact surface between GB chain and GR chain in the GB/GR and GB/GRM complexes. Proteins are displayed in lines. Contact surfaces were mapped and colored according to the distances between two chains.

(E) Average values of GABA I_max induced by 1 mM GABA in HEK-293 cells co-expressing GABA_ARs and various R271 site mutant GlyR α₁ subunits. All data were normalized to their respective controls (WT group).

(F) Correlation analysis of CoMSIA values of various amino acids at 271 and the percentage inhibition of GABA I_max.

(G) GlyRα₁ was purified using GABA_AR α₁ antibodies in HEK-293 cells co-expressing GABA_ARs (α₁β₂γ₂) and GlyRα₁ carrying various R271 mutations, and the co-precipitating proteins were detected by immunoblotting. Inputs are immunoblots of the same protein in cell lysates before Co-IP. Quantification of WT and R271 mutant GlyRα₁ binding to GABA_AR α₁ (n = 3). Data were normalized to the WT group.

(H) Correlation analysis of the percentage decrease in GABA I_max and amount of R271 mutant α₁ GlyRs co-immunoprecipitated with GABA_ARs.

All digits within the columns represent numbers of cells measured. Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001 based on unpaired t tests; ns, not significant (p > 0.05).