Cross-linking at the mid-point of the pore results in a channel “trapped” in its open S0 state.
A) Representative traces of the A14C-S193C-mVDAC1 gating activity at +40 mV, following CuX2Ph or DTT pre-treatment (materials and methods). Pre-treated with DTT and subsequently reconstituted A14C-S193C-mVDAC1 exhibited the expected native-like channel gating. The oxidized mutant however, appeared to remain constitutively open (92% of the collected traces) with relaxation in the closed S2 state being strongly disfavored (8% of the collected traces). B) Ohm-plot used for the determination of the conductances of the observed states in the gating transitions of the A14C-S193C-mVDAC1 channel. The open state (blue squares) conductance of this variant was 3.74±0.09 nS (n = 30) and was similar to that of the native protein (P = 0.30). When the cross-linked channel relaxed in a closed state (green squares) the determined conductance was 1.85±0.07 nS (n = 14), which was similar to the S2 state conductance of the native protein (P = 0.86). Bars represent standard errors of a minimum of 3 replicates per measurement. C) Average dwell times of native and oxidized A14C-S193C mVDAC1. Mean times are shown in grey (native channel; n = 310 and A14C-S193C oxidized variant; n = 14) with red bars indicating the standard error. The mean dwell time (30.61±6.39 min) for the oxidized mutant channel, before switching to a different state, was prolonged 180-fold relative to that of the native channel (0.17±0.03 min). T-tests indicated that the observed difference was statistically significant (P = <10−4). Values are given as mean ± SEM.