Radioligand binding

Protein expression was induced by addition of 5 mM sodium butyrate to the culture medium. After 16 h, the medium was removed and the cells were washed with PBS (calcium and magnesium-free) and detached. The cell suspension was collected, placed on ice and centrifuged for 5 min at 4°C and 1200 rpm in a Beckman GS6R centrifuge. After removal of the supernatant the cell pellet was washed and collected by re-suspension in PBS and centrifugation. The final pellet was weighed and frozen at -80°C until use.

Frozen pellets were thawed and homogenized in 10 volumes (w/v) of membrane preparation buffer (50 mM HEPES pH 7.4, 1 mM EDTA, 50 μg/mL bacitracin and protease inhibitors) using a Polytron Ultraturrax (twice for 15 s per cycle). The homogenate was centrifuged for 20 min at 4°C and 18500 rpm in a SL-50T Sorvall rotor and the pellet was re-suspended in membrane preparation buffer and re-homogenized as before. After centrifugation for 20 min at 4°C and 18500 rpm, the pellets were re-suspended in 5 volumes of membrane preparation buffer and divided into aliquots before freezing at -80°C. Protein concentration was determined using BioRad Protein Assay (Milan, Italy) with a BSA standard curve.

Stock solutions of test compounds (10 mM) were prepared in DMSO and stored at -20°C until use. Further dilutions were performed in DMSO to provide an 11-point concentration response curve spanning final concentrations from 0.01 nM to 10 μM. Radioligand binding experiments were performed immediately after transferring 2 μL of each concentration of test compound to a 96-well plate. Each well contained a final volume of 200 μL buffer (50 mM HEPES, 3 mM MnCl₂, 0.02% BSA, 0.02% Pluronic F-127 and 50 μg/mL bacitracin, pH 7.4). All reactions (except for [³H]-Septide saturation curve) were stopped by rapid filtration through Unifilter-96 GF/C filter plates pre-soaked for one hour in 0.5% PEI followed by 3 washings with 1 mL ice-cold 0.9% NaCl using a Packard cell harvester. After drying for 1 h at 40°C, 50 μL of Microscint-20 was added to each filter plate and bound radioactivity was measured using a Microplate TopCount (Packard C9912). [³H]-Septide saturation reactions were terminated by rapid filtration through GF/B filter paper pre-soaked in PEI 0.5% (w/v) solution and washed with 1 mL of ice cold 0.9% NaCl before filtration on a Brandel Harvester. Filters were washed 4 times with 1 mL ice cold 0.9% NaCl and placed into pico vials with 4 mL of Filter Count. The radioligand concentration was determined by measurement of 50 μL of [¹²⁵I]-NKA, or 100 μL of [³H]-Septide, mixed with 3 mL of Filter Count using a β-Counter TriCarb 2900.

100 μL of [¹²⁵I]-NKA (specific activity 81.4 TBq/mmol) was incubated with 100 μL of the CHO-hNK2 membrane suspension under the following conditions: to determine protein linearity, 0.1 nM [¹²⁵I]-NKA was incubated with increasing concentrations of CHO-hNK2 membranes (1, 3, 10 and 30 μg/well) at 23°C for 2 h; to examine association kinetics, 0.1 nM [¹²⁵I]-NKA was incubated with CHO-hNK2 membranes (6 μg/well) at 23°C for a range of durations from 10 to 240 min; in the saturation study, final concentrations of [¹²⁵I]-NKA and NKA from 0.02 to 5 nM (1 part hot/4 parts cold) were incubated with CHO-hNK2 membranes (6 μg/well) at 23°C for 3 h; in competition binding experiments, test compounds were incubated with 0.1 nM [¹²⁵I]-NKA and CHO-hNK2 membranes (6 μg/well) at 23°C for 3 h. Total binding was defined by the addition of 2 μL DMSO, and nonspecific binding was defined by the addition of 2 μL of 100 μM NKA (1 μM final concentration).

100 μL of [³H]-Septide (specific activity 3.9 TBq/mmol) was incubated with 100 μL of CHO-hNK1 membrane suspension under the following conditions: to determine protein linearity, 4 nM [³H]-Septide was incubated with increasing concentrations of CHO-hNK1 membranes (10, 15, 20, 25 and 30 μg/well) at 23°C for 90 min; to examine association kinetics, 5 nM [³H]-Septide was incubated with CHO-hNK1 membranes (17 μg/well) at 23°C for a range of durations from 2 to 120 min; in the saturation study, final concentrations of [³H]-Septide from 0.1 to 100 nM were incubated with CHO-hNK1 membranes (20 μg/well) at 23°C for 2 h; in competition binding experiments, test compounds were incubated with 5.0 nM [³H]-Septide and CHO-hNK1 membranes (20 μg/well) at 23°C for 1 h. In the saturation binding studies using different concentrations of [³H]-Septide, incubation for 2 h were carried out to allow all concentrations, including the lowest, to reach equilibrium. The association study with 5 nM [³H]-Septide confirmed that equilibrium was reached after 1 h and, therefore, 1 h was selected for the displacement binding studies. Total binding was defined by the addition of 2 μL of DMSO, and nonspecific binding was defined by the addition of 2 μL of 100 μM septide (1 μM final concentration).