Materials
Bovine serum albumin (BSA), EDTA, MgCl2, MnCl2, doxycycline, hygromycin B solution, methyl-β-cyclodextrin (MβCD), phenylmethylsulfonyl fluoride (PMSF), penicillin, streptomysin, gentamicin sulphate, serotonin and Tris were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM/F-12 [Dulbecco's modified Eagle's medium:nutrient mixture F-12 (Ham) (1:1)] and fetal calf serum (FCS) were obtained from Invitrogen/Life technologies (Grand Island, NY). Bicinchoninic acid (BCA) assay reagent was from Pierce (Rockford, IL). Amplex red cholesterol assay kit was purchased from Molecular Probes/Invitrogen (Eugene, OR). [3H]8-OH-DPAT (specific activity 141.1 Ci/mmol) was purchased from MP Biomedicals (Santa Ana, CA). GF/B glass microfiber filters were from Whatman international (Kent, U.K.). All other chemicals used were of the highest purity available. Water was purified through a Millipore (Bedford, MA) Milli-Q system and used throughout.
Cells and cell culture
Human embryonic kidney (HEK-293) cells stably expressing N-terminal myc-tagged wild-type human serotonin1A receptors (termed HEK-5-HT1AR cells) or K101A mutant were maintained in DMEM/F-12 (1:1) supplemented with 2.4 g/l of sodium bicarbonate, 10% (v/v) FCS, 60 μg/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml gentamicin sulfate and 250 μg/ml hygromycin B (complete media). Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. The cell culture medium was supplemented with 1 μg/ml doxycycline for 24 h for induction of receptor expression prior to experiments.
Membrane cholesterol depletion and estimation
Cholesterol was depleted from HEK-5-HT1AR wild-type and K101A mutant cells in an acute fashion using MβCD (Pucadyil et al. 2007). For this, cells were treated with 10 mM MβCD in serum-free medium for 30 min at 37 °C. Subsequently, to remove the serum-free media containing MβCD, cells were washed with PBS and harvested using ice-cold hypotonic buffer (10 mM Tris, 5 mM EDTA and 0.1 mM PMSF (pH 7.4)). Cholesterol content of cell membranes was estimated using the Amplex Red cholesterol assay kit (Amundson and Zhou 1999).
Cell membrane preparation
Cell membranes were isolated as described previously (Kalipatnapu et al. 2004). Briefly, cells were harvested in ice-cold hypotonic buffer and were homogenized for ~15 s with a polytron homogenizer at maximum speed. Following this, the cell lysate was centrifuged at 500 x g for 10 min at 4 °C. The post-nuclear supernatant was further centrifuged at 40,000 x g for 30 min at 4 °C and the final pellet containing membranes was resuspended in 50 mM Tris buffer (pH 7.4). The total protein concentration in isolated membranes was measured using the BCA assay (Smith et al. 1985).
Incubation of cell membranes at varying time periods
Membranes isolated from control and cholesterol-depleted cells expressing the wild-type or K101A mutant serotonin1A receptors were incubated at 37 °C for different time points ranging from 1 to 4 h. After incubation, radioligand binding assays were carried out at 25 °C.
Radioligand binding assay
Tubes in duplicate containing ~100 μg total membrane protein in a volume of 1 ml of buffer (50 mM Tris, 1 mM EDTA, 10 mM MgCl2, 5 mM MnCl2 (pH7.4)) were incubated with the radiolabeled specific agonist [3H]8-OH-DPAT for 1 h at 25 °C. Binding assays were performed using 1 nM radiolabeled agonist [3H]8-OH-DPAT. Nonspecific binding was measured by carrying out the binding assay in presence of 10 μM unlabeled serotonin. The binding reaction was terminated by rapid filtration under vacuum in a Millipore multiport filtration apparatus through Whatman GF/B filters (1.0 μm pore size) which were pre-soaked in 0.3% (w/v) polyethylenimine for 1 h (Bruns et al. 1983). Following this, filters were washed three times with 5 ml of ice-cold water, dried and the retained radioactivity was measured in a Packard Tri-Carb 2900 liquid scintillation counter (Perkin Elmer, Waltham, MA) using 5 ml of scintillation fluid.
Statistical analysis
Significance levels were evaluated using a Student's two-tailed unpaired t-test using GraphPad Prism software, version 4.0. Plots were generated using OriginPro 2022, version 9.9 (OriginLab, Northampton, MA).