NMDA Receptor Binding Studies

In vitro binding affinities (K_i) of the target compounds at the PCP site within the NMDAR channel were determined using competitive radioligand binding studies with [³H]-MK-801 in accordance with established protocols published by Reynolds and Sharma [22, 23]. Thoroughly washed rat forebrain homogenate were used as the NMDAR source (whole brain obtained from Pel-Freez Biologicals) and prepared as described by Reynolds and Sharma [22]. Suspensions of 10 mM HEPES buffer (pH 7.4 at room temperature) containing 100 μg/mL protein, 1 nM (+)-[³H]-MK-801, 100 μM glutamate, 10 μM glycine, and various concentrations of unlabeled competitor or 30 μM (+)-MK-801 for nonspecific binding (and positive control), were incubated in the dark on a mechanical rocker at room temperature for 2 h. The reaction was terminated by vacuum filtration using a 24 well cell harvester (Brandel, Gaithersburg, MD) over presoaked GF/B glass fiber filters (Brandel, Gaithersburg, MD). Filters were washed with room temperature assay buffer (3 x 5 mL). Tritium trapped on the filter was measured via liquid scintillation counting, using a Beckman LS 6500 multipurpose scintillation counter (BeckmanCoulter, USA) at 57% efficiency. IC₅₀ values were determined in Graphpad Prism 5.0 using non-linear regression with log-concentration plotted against percent specific binding. Percent specific binding for [³H]-MK-801 in a control experiment was ~95%. K_i values were calculated using the equation of Cheng and Prusoff [24]. The K_d for (+)-MK-801 (1.747 nM), was determined via homologous binding assay as described by Reynolds and Sharma and is consistent with the literature [22]. Protein concentration was determined via the Bradford method using Coomassie protein assay reagent (Sigma, USA) [25] with rat albumin (Sigma, USA) as standard. Experiments were performed in duplicate and repeated a minimum of three times. Raw counts per minute (CPM) for all NMDAR binding experiments are provided as supporting information (S1 File).