Effects of cytoplasmic [Cl⁻] on the current amplitudes of the O₁ and O₂ states in N306D. (A) Representative single-channel opening events of N306D under different intracellular chloride concentrations as marked with the corresponding all-points histograms on the right. The current amplitudes of both the O₁ state and O₂ state are increased with increasing chloride concentrations, but the difference between these two open states remains measureable even at the highest concentration tested (1,129 mM). (B) Summary of single-channel amplitudes of N306D at different cytoplasmic chloride concentrations. (C) Summary of normalized difference (Δi/i_O1, left vertical axis) and absolute difference (Δi, right vertical axis) between the O₁ and O₂ states under different chloride concentrations.