Figure 9: Biotinylation experiments.

(a) Effect of the MTSEA-B reagent and cholesterol depletion on [³H]ZM241385-specific binding (mean±s.e.m.) at a saturating radioligand concentration of 40 nM. (b) Scheme of cholesterol influence on receptor biotinylation according to the experimental results presented in a. Bar 1 (control, n=11): ZMA binding in untreated (cholesterol-containing) control cells yields low ZMA binding due to cholesterol shielding effect. Bar 2 (n=5): ZMA binding in MβCD-treated cell membranes (cholesterol-depleted) results in highest ZMA binding due to an empty binding pocket. Bar 3 (n=4): biotinylation of a cholesterol-depleted binding pocket results in a high degree of biotinylation in the orthosteric binding pocket. As the binding pocket is occupied by the biotinylated side chain of C3.30, ZMA binding is reduced. Bar 4 (n=4): low biotinylation degree due to the shielding effect of cholesterol in the binding pocket. Subsequent removal of the shielding agent cholesterol with MβCD results in an empty binding pocket that allows for high ZMA binding. Bar 5 (n=4): lowest ZMA binding due to the accumulated hindering effect of biotinylation and cholesterol. CHL, cholesterol (yellow); BIO, biotinylation (green); MβCD, methyl-β-cyclodextrin (blue); ZMA, ZM241385 ligand (red). In a **P<0.01 and ***P<0.001 significantly different from control 1, ^&P<0.05 significantly different between them (bars 3 and 4) according to a Student’s t-test.