Figure 6

Inhibition of BRET between Nluc and CA200645 at the A₃R by K5, K17, K18 and MRS 1220. HEK293 cells stably expressing Nluc-A₃R were treated with 5 nM CA200645 and increasing concentrations of unlabelled compound (represented in nM) (A) K5, (B) K17, (C) K18 or (D) MRS 1220. For MRS 1220, this trace demonstrates a classic tracer ‘overshoot’, as has been previously described observed when the unlabelled compound has a slower off rate than the labelled CA200645 (K_off of 0.0248 ± 0.005 min⁻¹ and 0.4397 ± 0.014 min⁻¹ respectively) (Sykes et al.²⁴, Motulsky and Mahan²⁸). The data shown are representative of three independent experimental repeats (mean ± SEM) fitted with the appropriate model, as determined by statistical comparison between our new model (“Kinetics of competitive binding, rapid competitor dissociation”, derived in the Appendix I) (K5, K17 and K18) or the ‘kinetic of competitive binding’ model (built into Prism) for MRS 1220 (see “Materials and methods” for fitting procedure and statistical comparison method). (E) The resulting concentration dependent decrease in BRET ratio at 10 min was taken to calculate pK_i through fitting the Cheng-Prusoff equation⁵⁹. Each data point represents mean ± SEM of five experiments performed in duplicate.