Intrinsic gating of human AChRs. (a) Left: Mutations far from the binding sites produce similar changes in the diliganded gating energy with Cho (ΔΔG₂^Cho) in mouse and human AChRs (slope, 1.0 ± 0.1; R² = 0.95). Each symbol is a different mutation. Right: In adult-type human AChRs, ΔΔG₂^Cho is caused exclusively by a change in the unliganded gating energy (ΔG₀^obs; slope = 1.0 ± 0.1, R² = 0.91; dashed lines, 95% confidence limits). The y intercept (no change in ΔΔG₂^Cho) is ΔG₀ in the WT. Inset: Voltage dependence of E₀ in adult-type human AChRs. (b) Example unliganded single-channel current clusters from mutations added to four different background constructs. The clusters (top to bottom) and the backgrounds (left to right) are arranged with increasing open-channel probability (excluding long openings).