Figure 6.

The SSCs do not involve the pores per se. (A) Plot of conductance ratios versus PEG molecular weight. The conductances were obtained from three or more independent experiments in patch-clamp (150 mM KCl; pH 7.2), and normalized to the average conductance measured for a fully excluded PEG (PEG₄₆₀₀) to control for potential artifacts due to the presence of PEGs in the pipette. Experiments were carried out in patch-clamp to prevent insertions of monomers, which often occur in bilayers when PEGs are present. Dark circles represent the ratio of the left-over conductance during an SSC, and gray circles are the ratio of the fully open trimer conductance. The dotted line represents the fit obtained from bilayer data on OmpU from Fig. 2B, normalized to the excluded-PEG conductance ratio. (B) Plot of the average dwell time of the SSCs (average t_c) in the presence or absence of nonpenetrating PEG4600 in buffer with 150 mM KCl (pH 7.2) at a pipette voltage of +50 mV. The error bars are SE (n ≥ 3). Whether PEG4600 is present on both sides of the patch, or only on the pipette side or the bath side is indicated. (C) Patch-clamp trace of a single trimer of OmpU obtained in the presence of symmetric PEG100 in 150 mM KCl buffer at pH 6.2. The cumulative effect of PEG100 and pH 6.2 leads to a stable SSC state (current level at −90 mV marked by letter a). The voltage was then switched to −190 mV in the hope of reopening the channel, but this did not occur and the channel remained in the SSC state (current level at −190 mV marked by letter b). The high voltage, however, triggered the voltage-dependent inactivation of the porin (13) in three consecutive steps representing the sequential closure of each pore of the trimer.