Figure 14

Ligand depletion improves precision of estimated K _i2 for nicotine with noisy data and NSB. Ligand depletion improves precision of estimated K_i2 for nicotine with noisy data. A and B. Increasing concentrations of binding sites and [³H]EB samples a wide range of fractional contributions of the two binding sites and NSB to total [³H]EB binding as nicotine concentration varies. In A, the combination [³H]EB = 0.013 nM and R1T = 0.13 nM predominantly samples interaction between [³H]EB and nicotine at the high affinity site. In B, the combination [³H]EB = 20 nM and R1T = 20 nM with substantial ligand depletion more effectively samples interaction between [³H]EB and nicotine at the low affinity site. The low affinity site contributes a maximum of one-tenth of total [³H]EB binding and contributes more than NSB does to total [³H]EB binding up to about 1000 nM nicotine. The y-axis values were calculated as Q/(R1L+R2L+NSB) where Q = R1L, R2L, or NSB. C. Noisy heterologous competition data for [³H]EB and nicotine with various maximum S/N were fit with two sites_total model to estimate K_i1 and K_i2. The K_i2 estimates shown with green Δ were derived with R1T = 0.13 nM. Modest ligand depletion and negligible occupancy by [³H]EB of the low affinity binding site lead to low precision of K_i2 estimates at maximum S/N = 300. K_i2 estimates shown with blue ▲ were derived with R1T = 0.13, 1, 20 nM. Increasing ligand depletion and occupancy of the low affinity site by [³H]EB lead to more precise K_i2 estimates with noisier data. Error bars show standard deviations. K_i1 estimates (green ○ or blue ●) are relatively independent of maximum S/N values. The number of data points (114 points) was identical in the two sets of estimates. Solid line: true K_i1 (0.84 nM); dashed line: true K_i2 (775 nM); α_L = 0.1; α_B = 0.