OccK Channels fromPseudomonas aeruginosaExhibit 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 earlier expectations, 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 (OccK3)-, two (OccK4, OccK5 and OccK6)- and three (OccK1, OccK2 and OccK7)-open sub-state 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.

Toward Understanding the Outer Membrane Uptake of Small Molecules by Pseudomonas aeruginosa

“…An interesting property of many Occ proteins, including OccK2, is their dynamic behavior in single channel conductance experiments (18,50,51). Although the conductance of the most dominant state of OccK2 is relatively large for a substrate-specific channel (ϳ230 pS in 1 M KCl), there are frequent downward conductance spikes that occasionally close the channel completely (18).…”

The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity

“…In recent years, molecular modeling and electrophysiology studies revealed the ability to inhibit the protective antigen of anthrax toxin by small-molecule, high-affinity inhibitors. 53, 54 This approach might prove more effective than that of developing new antibiotics against P. aeruginosa , requiring their navigation through the outer membrane carboxylate channels OccD 55 and OccK, 56, 57 because of their relatively small internal diameter. 58, 59 A more efficacious approach is to target the transport of capsular polysaccharides across the outer membrane of Gram-negative bacteria.…”

Theoretical analysis of ion conductance and gating transitions in the OpdK (OccK1) channel

“…20 Though details vary, this kind of gating behavior is seen independent of salt concentration and applied voltage. 9 Cheneke et al 5 experimentally studied different loop deletions to understand the dynamics of the single channel current fluctuations exhibited by the native OpdK channel.…”

“…20 However, the nature of the underlying gating mechanism and the transition pathway are unknown. In a series of papers 5,9,18,20 the subconduction states of OpdK have been detailed with respect to varying transmembrane voltage, ion concentration and temperature. A non-trivial dependence on these parameters has been found and model explanations have been provided.…”