N306C/Cysless modified by MTSES⁻ shows O₁ to O₂ transitions. Single-channel recordings were conducted under −50 mV. (A) The left panel shows a representative single-channel trace of N306C/Cysless, and the all-points histogram on the right side shows only one conductance level (0.40 ± 0.02 pA, n = 14). (B) Single-channel recordings of N306C/Cysless modified by negatively charged thiol-specific reagent MTSES⁻ show two distinct single-channel conductance levels: a small one, O₁ (0.19 ± 0.02, n = 3), and a large one, O₂ (0.30 ± 0.02, n = 3), with a preferred transition order of C→O₁→O₂→C. The difference between two open-channel conductance levels is clearly differentiated in the all-points histogram on the right. (C) In contrast, N306C/Cysless modified by positively charged thiol-specific reagent MTSET⁺ shows a single and yet larger open-channel current amplitude (0.49 ± 0.02, n = 4) compared with the one before modification in A. (D) A representative recording of the washout phase of a macroscopic current trace using ATP-free perfusion solution from a patch containing multiple MTSES⁻-modified N306C/Cysless channels locked open by PP_i and ATP. The stepwise closing of each channel is shown in the trace on the left, and the long burst duration in the absence of ATP before channels close is indicative of these channels being locked open by PP_i and ATP into an open-channel conformation with two NBDs in a head-to-tail dimeric configuration. The single-channel amplitude (0.19 ± 0.01, n = 4) measured in the all-points histogram on the right is identical to the single-channel amplitude of the O₁ state in B.