Fig. 4: Conformation-dependent allosteric binding sites of cholesterol.

a Cholesterol binding sites depicted in the occluded state. The left panel shows the overall structure of cholesterol binding sites, with GlyT1 and cholesterol molecules represented as cylinders and spheres, respectively. b Representative cholesterol binding sites and surface electrostatic potential maps of GlyT1 in outward-facing (GlyT1^SSR), occluded (GlyT1^Sar), and inward-facing (GlyT1^ICL) states. Cholesterol is shown as yellow spheres and labeled. c–e Structural comparison of cholesterol binding sites between occluded (GlyT1^Sar, light blue) and outward-facing (GlyT1^SSR, grey) states. Red arrows indicate helix and residue movements during conformational transitions for (d) CHOL1 and (e) CHOL2. Residues involved in the interactions are shown as sticks and labeled. f, g Effects of cholesterol depletion on GlyT1 functional activity in HEK−293F cells. Cholesterol concentrations in cells after brief incubation with MβCD (0, 2.5, or 3.5 mg/mL) are shown. [³H]Glycine uptake plots for GlyT1 in the absence (0 mg/mL, blue) or presence (2.5 mg/mL, red; 3.5 mg/mL, orange) of MβCD. No significant differences in K_m values compared to WT GlyT1 (P = 0.4221 for 2.5 mg/mL, P = 0.1735 for 3.5 mg/mL). V_max values show significant differences in presence of 2.5 mg/mL (P < 0.0001) and 3.5 mg/mL (P < 0.0001) MβCD. V_max values for 2.5 mg/mL and 3.5 mg/mL MβCD also differ significantly (P = 0.0172). Data from three independent experiments presented as mean ± SEM. Statistical comparisons were conducted using one-way ANOVA followed by an unpaired t-test to determine significant differences. h Cell surface biotinylation of WT GlyT1 incubated with 0, 2.5, and 3.5 mg/mL MβCD in HEK293F cells. Surface proteins (lanes 1–3) labeled with biotin and recovered using streptavidin beads. Biotinylated Na⁺/K⁺ ATPase served as loading control. Experiments repeated three times with similar results. i Densitometric quantification of surface expression for WT GlyT1 incubated with 0, 2.5, and 3.5 mg/mL MβCD in HEK293F cells. Gels that derived from the same experiment were processed in parallel. The surface expression was first normalized to Na⁺/K⁺ ATPase and then to the expression of WT GlyT1. Each black dot, square, and triangle symbol represents an individual data point for WT GlyT1 incubated with 0, 2.5, and 3.5 mg/mL MβCD, respectively. Data are shown as mean ± S.E.M. (n = 3 biologically independent experiments).