N306D/E channels present a clear O₁O₂ phenotype. (A) A representative single-channel recording of N306D in the WT background shows the characteristic O₁O₂ phenotype, supporting the idea that it is the negative charge introduced at position 306 that accounts for the O₁O₂ phenotype displayed in N306D and in MTSES⁻-modified N306C/Cysless. Single-channel amplitudes of O₁ (0.18 ± 0.01, n = 3) and O₂ (0.24 ± 0.01, n = 3) states can be measured in the all-points histogram on the right. (B) Expanded opening bursts show details of each opening events as marked in A. (C) A representative single-channel recording of N306E in the WT background further confirms the role of a negative charge at position 306 in conferring the O₁O₂ phenotype. The all-points histogram shows distinct single-channel amplitudes of the O₁ (0.19 ± 0.01, n = 3) and O₂ states (0.27 ± 0.01, n = 3).