Fig. 2.

Characterization of ³H-CHIBA-3007. (A) Ion dependence of [³H]CHIBA-3007 binding. CHIBA binding was measured by incubating membrane fractions prepared from the cells expressing WT hGlyT1b with [³H]CHIBA-3007 in binding buffer with or without Na⁺, Cl⁻, or both. Equimolar NMDG⁺ and gluconate were used to replace Na⁺ and Cl⁻, respectively. In experiments measuring displacement of [³H]CHIBA-3007 with unlabeled CHIBA-3007, membranes were incubated with radiolabeled CHIBA-3007 (kept at 1 nM) together with unlabeled CHIBA-3007 at 0 to 1,000 nM. The graph shows a representative experiment, with binding expressed as a percentage of that measured in the absence of unlabeled CHIBA-3007. All error bars shown in the figure represent SDs from triplicate measurements. The K_D values for CHIBA-3007 were estimated to be 140.9 ± 1.8 nM in the absence of Na⁺ and Cl⁻ (control), 141 ± 10 nM (Cl⁻ alone), 42 ± 5.6 (Na⁺ alone), and 4.9 ± 0.5 nM (NaCl), respectively. These calculated values represent the mean ± SEM of three experiments with triplicate measurements. (Inset) Ion dependence of 1 nM [³H]CHIBA-3007 binding to WT GlyT1b membranes. Binding, expressed per mg of membrane protein, was corrected for nonspecific binding using membranes from untransfected cells. Error bars represent SDs from triplicate measurements. (B) Glycine displacement of CHIBA-3007 binding. Glycine was added at final concentrations from 0 to 1,000 mM along with [³H]CHIBA-3007 (1 nM). The graph shows a representative experiment. All error bars shown in the figure represent SDs from triplicate measurements. At concentrations up to 1 M, glycine displaced less than 5% of CHIBA-3007 binding in the absence of Na⁺. The estimated K_I values for displacement by glycine were >1 M with Na⁺ alone and 0.33 ± 0.01 mM in NaCl. This calculated value represents the mean ± SEM of three experiments, each with triplicate measurements.