Estimating E₀^wt using mutations that increase the gating equilibrium constant. There was no agonist present in the pipette or bath. (A) Low time-resolution view of clusters of unliganded openings (open is down; −100 mV). Each cluster reflects the constitutive gating activity of an individual AChR. The background was βT456I + εE181T + εL269F (Table S1). (B) Estimation of the unliganded gating equilibrium constant of wt AChRs (E₀^wt) from different mutant sets (1, open; 2, closed). The data from set 1 are from Purohit and Auerbach (2009). The x-axis value is the log of the product of the fold increases in E₂^Cho for each mutation alone (Table S1), and the y axis is the log of E₀^obs measured directly from the clusters. For both mutant sets, the extrapolated ordinal intercept was approximately −6.12 (unweighted straight-line fit shown with 95% confidence intervals), which corresponds to E₀^wt = ∼7.6 × 10⁻⁷. (Inset) Higher time-resolution view of clusters from mutant set 2 (αA96) expressed on the same background.