Fig. 4.

Displacement of tritium-labeled PbTx-3 ([³H]PbTx-3) from rat brain synaptosomes. Binding experiments were performed as described previously (Poli et al., 1986). Briefly, synaptosomes were prepared from frozen whole rat brains (male, Sprague–Dawley) by homogenization in 0.32-M sucrose (containing protease inhibitors: 1-mM iodoacetamine, 1 mM 1,10-phenanthroline, 0.1-mM phenylmethylsulfonyl fluoride, 1-μM pepstatin A) followed by three centrifugation steps. The P3 fraction was diluted to 1 mg/mL protein (determined by a modified Lowry technique) with HEPES binding medium containing protease inhibitors. Binding was determined at ice temperature in 1 mL total volume in the presence of 1 mg/mL bovine serum albumin. Serial dilutions of competing ligands were prepared in ethanol and added (10 μL) to 790 μL of binding medium in 1.5-mL Eppendorf centrifuge tubes (triplicates at each concentration of competitor). A solution of [³H]PbTx-3 in binding medium (100 μL) was added to yield a final concentration of approximately 5 nM, near the reported K_D value for rat brain synaptosomes (2.2 nM). After addition of 100 μL of synaptosome suspension, the contents of each tube were vortexed and allowed to equilibrate for >1 hr on ice. Tubes were then centrifuged, supernatant was quickly aspirated, the pellet washed with immediate aspiration, and the bottom of the tube (containing the pellet) removed. The radioactivity associated with the pellet was quantified by liquid scintillation spectroscopy. Nonlinear regression analysis was performed on the resulting competition binding data using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, California, www.graphpad.com). All derived values are the mean of at least three experiments, with triplicate determinations at each concentration of competitor in each experiment. a. A displacement curve for PbTx- is provided for reference. b. Binding experiments were performed as described above at four concentrations of [³H]PbTx-3 (in nM: 0.1, 1.5, 3, and 6). Radioactivity associated with the pellet (bound) and the supernatant (free) was determined. Linear regression analysis of each of the double reciprocal plots yielded r² values > 0.98. Convergence of the plots near the ordinate indicates that the interaction between the ligands is competitive. K_i for brevenal is 685 nM.