[35S]-GTPγS binding experiments
[35S]-GTPγS experiments were performed on crude membrane fractions of MOR-expressing HEK293 cells to determine mu-agonist-induced G-protein activation (as reflected by the exchange rate of GDP for GTP) at different pH values (6.5-7.4). GTP was replaced by a high concentration of [35S]-GTPγS in the assay solvent, and the accumulation of [35S]-GTPγS-bound G proteins in the membrane was measured. To this end, membranes were homogenized and dissolved in HEM G-protein buffer at pH 7.4 or 6.5, including freshly added 0.1 % (w/v) BSA and 1 mM dithiotreitol (DTT). In analogy to (Ludwig et al., 2003), 50 µg of membrane fractions in duplicates were incubated with GDP (30 µM) and [35S]-GTPγS (0.05 nM) for 90 min at 30oC to determine dose-response curves and EC50 values. Unspecific [35S]-GTPγS binding in the presence of non-radioactive GTPγS (10 µM) was subtracted to yield specific binding. Bound and free ligands were separated by rapid filtration under vacuum through Whatman GF/B glass fibre filters soaked in water followed by 6 washes with Tris Buffer. Bound radioactivity was determined by liquid scintillation spectrophotometry for 35S after overnight extraction of the filters in scintillation fluidoptiphase HISAFE3 . Concentrations of radioactive compound were calculated based on the half-life of 35S (87.4 days). Experiments were randomized to compensate for position effects in the filter apparatus or unequal sample processing times. Data processing and analysis were blinded for pH values (6.5 or 7.4) with the help of a colleague.