Size and dynamics of the Vibrio cholerae porins OmpU and OmpT probed by polymer exclusion

Abstract

The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.

Figure 1

Typical behavior of OmpU and OmpT at neutral and acidic pHs. Representative patch-clamp recordings of a (A and B) single OmpT trimer and (C–E) a single OmpU trimer in symmetric conditions of 150 mM KCl in buffer at the indicated pHs. The pipette voltage is +50 mV. The tick mark labeled “open” indicates the current level of the fully open trimer; C1, C2, and C3 indicate the levels corresponding to the closures OmpT monomers (A and B). (C–E) Tick mark of labeled SSC indicate the single step closure level observed for OmpU. The dashed line is the 0 pA level. Note the increase in the size of the SSC whereas the full conductance of the open trimer does not vary.

Figure 2

OmpU has a larger effective radius than OmpT. (A) Effect of penetrating PEGs on the OmpU trimer. The two traces were obtained from the same inserted trimer in planar lipid bilayer (150 mM KCl; pH 7.2), before and after adding PEG200 at a final concentration of 10.9% (w/w). The voltage was stepped from 0 to +70 mV. Note the decrease in the current level corresponding to the fully open trimer (level marked open). (B) Molecular weight dependence of the conductance ratio. The conductance ratio is the ratio of the fully open trimer conductance measured in the presence of PEG over the conductance measured without PEGs in the same bilayer. Each data point represents the average conductance ratio measured on two (no error bar) or three or more (error bar) independent experiments. The error bars represent the SE. The data obtained for OmpU (solid circles) and OmpT (open circles) were fitted to Eq. 1 in Materials and Methods. The molecular weight cut-offs (ω₀) were found to be 916 ± 91 and 598 ± 65 for OmpU and OmpT, respectively. The corresponding effective radius for OmpU and OmpT are 0.55 nm and 0.43 nm, respectively. Other parameters obtained from the fits were: for OmpU: χ = 0.31 ± 0.03; α = 1.59 ± 0.36; (conductance ratio)_max = 1.03 ± 0.01; and for OmpT: χ = 0.32 ± 0.03; α = 1.21 ± 0.24; (conductance ratio)_max = 1.03 ± 0.01.

Figure 3

The effective radius of OmpU is increased at acidic pH. The conductance of the fully open OmpU trimer was measured in patch-clamp at different symmetric pHs, and in the absence or the presence of a penetrating PEG (PEG600) or a nonpenetrating PEG (PEG4600) applied symmetrically. Each conductance measurement was derived from the current voltage relationship. In the absence of PEGs, the conductance of OmpU is not significantly modified by acidic conditions. The presence of penetrating PEG600 decreases the conductance to a larger extent at lower pH; at pH 5.7, g₆₀₀/g₄₆₀₀ = 0.69, which is the lower limit due to the effect of PEGs on conductivity, suggesting that PEG600 can fully enter in the pore.

Figure 4

Higher ionic strength stabilizes the SSCs. (A–D) Representative current traces of OmpU recorded at various symmetric KCl concentrations (pH 7.2), as indicated. The open trimer conductance is increased by higher ionic strength as indicated by the change in scale bar from A to D. The pipette voltage was +50 mV. Note that the SSCs become larger, longer in duration and more frequent as the buffer ionic strength is increased. (A,C,D) Recorded in planar lipid bilayer. (B) Recorded in patch-clamp. (E) Plot of the average ratio of SSC conductance (g(SSC)) over the open trimer conductance (g(trimer)) versus KCl concentration in the buffer. Error bars are SE (n ≥ 3). (F) Plot of the average dwell time of the SSC (average t_c; black histogram bars) and of the dwell time of the fully open trimer (average t_o; white histogram bars) versus KCl concentration in the buffer (at pipette voltage of +50 mV). Error bars are SE (n ≥ 3). Note the logarithmic scale for the left y axis.

Figure 5

The presence of PEGs promotes SSCs. (A–C) Representative traces of a single OmpU trimer were recorded in patch-clamp at +50 mV in the (A) absence or the (B) presence of symmetric penetrating PEG600 or (C) nonpenetrating PEG4600 in a 150 mM KCl buffer at pH 7.2. The presence of PEGs on both sides of the membrane triggers longer and more frequent closures. (D) Plot of the average dwell time of the SSC (average t_c; black histogram bars) and of the dwell time of the fully open trimer (average t_o; white histogram bars) in the absence or the presence of PEGs, as indicated. Error bars are SE (n ≥ 3). Pipette voltage is +50 mV.

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.