Ligand depletion improves precision of estimated K
for nicotine with noisy data and NSB. Ligand depletion improves precision of estimated Ki2 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 sitestotal model to estimate Ki1 and Ki2. The Ki2 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 Ki2 estimates at maximum S/N = 300. Ki2 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 Ki2 estimates with noisier data. Error bars show standard deviations. Ki1 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 Ki1 (0.84 nM); dashed line: true Ki2 (775 nM); αL = 0.1; αB = 0.