OccK channels from Pseudomonas aeruginosa exhibit diverse single-channel electrical signatures but conserved anion selectivity

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium that utilizes substrate-specific outer membrane (OM) proteins for the uptake of small, water-soluble nutrients employed in the growth and function of the cell. In this paper, we present for the first time a comprehensive single-channel examination of seven members of the OM carboxylate channel K (OccK) subfamily. Recent biochemical, functional, and structural characterization of the OccK proteins revealed their common features, such as a closely related, monomeric, 18-stranded β-barrel conformation with a kidney-shaped transmembrane pore and the presence of a basic ladder within the channel lumen. Here, we report that the OccK proteins exhibited fairly distinct unitary conductance values, in a much broader range than previously expected, which includes low (~40-100 pS) and medium (~100-380 pS) conductance. These proteins showed diverse single-channel dynamics of current gating transitions, revealing one-open substate (OccK3), two-open substate (OccK4-OccK6), and three-open substate (OccK1, OccK2, and OccK7) kinetics with functionally distinct conformations. Interestingly, we discovered that anion selectivity is a conserved trait among the members of the OccK subfamily, confirming the presence of a net pool of positively charged residues within their central constriction. Moreover, these results are in accord with an increased specificity and selectivity of these protein channels for negatively charged, carboxylate-containing substrates. Our findings might ignite future functional examinations and full atomistic computational studies for unraveling a mechanistic understanding of the passage of small molecules across the lumen of substrate-specific, β-barrel OM proteins.

Figure 1. Structural representation of OccK1 family protein channels

(A) The molecular surface and ribbon representations of six OccK subfamily members whose crystal structures have been solved recently (11), which include OccK1, OccK2, OccK3, OccK4, OccK5, and OccK6. On the molecular surface representation, the side chains of positively charged amino acids are marked blue, and those of negatively charged amino acids are marked red. On the ribbon representation, constriction-forming loops L3, L4, L7 are colored in green, yellow, and orange, respectively; (B) The side view of the OccK5 channel in a planar lipid bilayer. The charged amino acids are colored as described above. Note the positive surface resulting from the presence of the basic amino acid ladder within the channel constriction.

Figure 2. Typical single-channel electrical traces of the OccK subfamily members and the corresponding all-points current amplitude histograms

Traces (the left panels) were recorded at a transmembrane potential of +60 mV, and in 1M KCl, 10 mM phosphate, pH 7.4. (A) OccK1; (B) OccK2; (C) OccK3; (D) OccK4; (E) OccK5; (F) OccK6; (G) OccK7. All-points current amplitude histograms (the right panels) included all acquired data points in a single-channel electrical trace. Current levels were marked on both the single-channel electrical traces and on the all-points current amplitude histograms. The sub-states O₁, O₂, and O₃ (if applicable) were assigned to the current levels from the lowest to the highest conductance values, respectively. The all-points current amplitude histograms were determined from single experiments, whose electrical traces are shown on the left panels. In this figure, the single-channel electrical traces corresponding to OccK1, OccK2, OccK3, OccK5 and OccK7 were low-pass Bessel filtered at 2 kHz. Those single-channel electrical traces corresponding to OccK4 and OccK6 were low-pass Bessel filtered at 10 kHz.

Figure 2. Typical single-channel electrical traces of the OccK subfamily members and the corresponding all-points current amplitude histograms

Traces (the left panels) were recorded at a transmembrane potential of +60 mV, and in 1M KCl, 10 mM phosphate, pH 7.4. (A) OccK1; (B) OccK2; (C) OccK3; (D) OccK4; (E) OccK5; (F) OccK6; (G) OccK7. All-points current amplitude histograms (the right panels) included all acquired data points in a single-channel electrical trace. Current levels were marked on both the single-channel electrical traces and on the all-points current amplitude histograms. The sub-states O₁, O₂, and O₃ (if applicable) were assigned to the current levels from the lowest to the highest conductance values, respectively. The all-points current amplitude histograms were determined from single experiments, whose electrical traces are shown on the left panels. In this figure, the single-channel electrical traces corresponding to OccK1, OccK2, OccK3, OccK5 and OccK7 were low-pass Bessel filtered at 2 kHz. Those single-channel electrical traces corresponding to OccK4 and OccK6 were low-pass Bessel filtered at 10 kHz.

Figure 2. Typical single-channel electrical traces of the OccK subfamily members and the corresponding all-points current amplitude histograms

Traces (the left panels) were recorded at a transmembrane potential of +60 mV, and in 1M KCl, 10 mM phosphate, pH 7.4. (A) OccK1; (B) OccK2; (C) OccK3; (D) OccK4; (E) OccK5; (F) OccK6; (G) OccK7. All-points current amplitude histograms (the right panels) included all acquired data points in a single-channel electrical trace. Current levels were marked on both the single-channel electrical traces and on the all-points current amplitude histograms. The sub-states O₁, O₂, and O₃ (if applicable) were assigned to the current levels from the lowest to the highest conductance values, respectively. The all-points current amplitude histograms were determined from single experiments, whose electrical traces are shown on the left panels. In this figure, the single-channel electrical traces corresponding to OccK1, OccK2, OccK3, OccK5 and OccK7 were low-pass Bessel filtered at 2 kHz. Those single-channel electrical traces corresponding to OccK4 and OccK6 were low-pass Bessel filtered at 10 kHz.

Figure 3. The I–V profiles of all open sub-states of the OccK subfamily members in 1M KCl, 10 mM potassium phosphate, pH 7.4

(A) OccK1; (B) OccK2; (C) OccK3; (D) OccK4; (E) OccK5; (F) OccK6; (G) OccK7; (H) The plots represent the conductance levels of each open sub-state. The big squares indicate the conductance value corresponding to the most probable open sub-state of each channel. The small dots denote the conductance values corresponding to the low-probability open sub-states of each channel. These conductance values are also shown in Table 1.

Figure 3. The I–V profiles of all open sub-states of the OccK subfamily members in 1M KCl, 10 mM potassium phosphate, pH 7.4

(A) OccK1; (B) OccK2; (C) OccK3; (D) OccK4; (E) OccK5; (F) OccK6; (G) OccK7; (H) The plots represent the conductance levels of each open sub-state. The big squares indicate the conductance value corresponding to the most probable open sub-state of each channel. The small dots denote the conductance values corresponding to the low-probability open sub-states of each channel. These conductance values are also shown in Table 1.

Figure 4. The free energy landscapes of the current gating transitions found in OccK proteins

(A) The free energy landscape for the two-open sub-state kinetic model, in which the O₁ level is assigned to the most probable open sub-state; (B) The free energy landscape for the three-open sub-state kinetic model, in which the O₂ level is assigned to the most probable open sub-state.

Figure 5. The kinetic rate constants of the current gating transitions and their corresponding free energy differences as well as estimated activation free energies

(A) OccK2; (B) OccK4; (C) OccK5; (D) OccK6; (E) OccK7. The activation free energies were estimated using a frequency factor of 10⁻⁶ s⁻¹ (30). The left y axes are the free energies in RT units, whereas the right y axes are the corresponding kinetic rate constants. Note that the kinetic rate constant axis is in a log scale, and the positive direction is downwards.

Figure 6. The voltage-dependent free energy landscapes of the low-conductance OccK4 and OccK6 channels

(A) Voltage dependence of the free energy landscape of the OccK4 channel; (B) Voltage dependence of the free energy landscape of the OccK6 channel.

Figure 7. The I–V profiles of the OccK1 channel under asymmetric salt concentrations

(A) pH 6.0; (B) pH 8.0. The corresponding numerical results pertinent to the ion selectivity features of OccK1 are shown in Table 2. The buffer solution contained 10 mM potassium phosphate.