NSB of a competitor changes K
but not K
. A. The two sites modelfree generated competition data for [3H]EB and nicotine with R1T = 0.013 nM and R2T = 3.9 nM so the competition curve clearly shows the two binding sites. Increasing αcompetitor shifted the curve rightward without changing the shape of the curve. B. To determine how increasing αcompetitor affected apparent values of Ki1 (Ki1, app) and Ki2 (Ki2, app) with αcompetitor, app = 0, two sites modeltotal was fitted to the competition curves in A. The only degrees of freedom for this fitting were Ki1, app and Ki2, app for nicotine. Increasing αcompetitor increases Ki1, app and Ki2, app but does not change Ki2, app/Ki1, app, a ratio reflecting the difference in free energy of nicotine binding at the two sites. C. Similar to A, the two sites modelfree generated competition data for [3H]EB and a superhigh affinity competitor with R1T = 0.013 nM, R2T = 3.9 nM, Ki1 = 0.00013 nM, and Ki2 = 0.12 nM. The curves clearly show the two binding sites. In contrast to A, shapes of the competition curves change as increasing αcompetitor shifts the curve rightward. D. Similar to nicotine, Ki2, app/Ki1, app for the superhigh affinity competitor does not vary with αcompetitor.