Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of Bacterial β-Barrel Channels

Abstract

The outer membrane of Gram-negative bacteria contains β-barrel proteins that form high-conducting ion channels providing a path for hydrophilic molecules, including antibiotics. Traditionally, these proteins have been considered to exist only in an open state so that regulation of outer membrane permeability was accomplished via protein expression. However, electrophysiological recordings show that β-barrel channels respond to transmembrane voltages by characteristically switching from a high-conducting, open state, to a so-called 'closed' state, with reduced permeability and possibly exclusion of large metabolites. Here, we use the bacterial porin OmpF from E. coli as a model system to gain insight on the control of outer membrane permeability by bacterial porins through the modulation of their open state. Using planar bilayer electrophysiology, we perform an extensive study of the role of membrane lipids in the OmpF channel closure by voltage. We pay attention not only to the effects of charges in the hydrophilic lipid heads but also to the contribution of the hydrophobic tails in the lipid-protein interactions. Our results show that gating kinetics is governed by lipid characteristics so that each stage of a sequential closure is different from the previous one, probably because of intra- or intermonomeric rearrangements.

Keywords: bacterial porins; beta-barrel channel; hydrophobic acyl chains; lipid headgroup charge; phospholipids; voltage gating.

Figure 1

Three-dimensional structure of the OmpF (Outer membrane protein F) channel. View from the extracellular side (left panel) and side view (right panel) of the OmpF homotrimer (PDB code 2OMF). Each monomer is folded as a 16-stranded antiparallel β-barrel connected by eight long loops facing the extracellular side and eight short turns facing the intermembrane space. Loop 3 (shown in black) folds inside the barrel, creating a constriction zone (~1 nm) at half the length of the monomer.

Figure 2

OmpF sequential closing depends on lipid headgroup charge. Representative current traces of a single OmpF trimer inserted in a neutral diphytanoyl-phosphatidylcholine (DPhPC, upper panels) or a negatively charged diphytanoyl-phosphatidylserine (DPhPS, lower panels) membrane, with the typical step-wise transitions after a high positive (a) or negative (b) voltage is applied. Next to each trace, the amplitude distributions are shown for each state (bar graph) with a Gaussian fitting (solid lines).

Figure 3

A negatively charged membrane accelerates closure of first OmpF monomer. Logarithmically binned histograms of the time the channel spends in the open conformation under a positive (a) or a negative (b) applied voltage when inserted in a neutral or negatively charge membrane, as indicated. Solid lines are exponential fittings. The characteristic times obtained from exponential fittings are shown in (c). See Figure S1 for the distributions in (a) and (b) displayed individually. In (c), significance between mean closure times was determined with a one-way ANOVA significance test. A Holm–Sidak post hoc test was used for pair-wise comparison (**: p < 0.01). See Materials and Methods for more details.

Figure 4

Change in acyl chain composition modulates closure of first OmpF monomer. Logarithmically binned histograms of the time the channel spends in the open conformation under a positive (a) or a negative (b) applied voltage when inserted in membranes composed of pure DPhPC or DPhPC/DOPC (diphytanoyl-phosphatidylcholine/dioleoyl-phosphatidylcholine 1/1, labelled as DOPC), as indicated. Solid lines are exponential fittings. The characteristic times obtained from exponential fittings are shown in (c). See Figure S1 for the distributions in (a) and (b) displayed individually. In (c), significance between mean closure times was determined with a one-way ANOVA significance test. A Holm-Sidak post hoc test was used for pair-wise comparison (NS (not significant): p > 0.2; **: p < 0.01). See Materials and Methods for more details.

Figure 5

Gating kinetics for the second and third monomer closures display higher complexity than the first one. Logarithmically binned histograms of the time the channel spends before closing the first (τ₀), second (τ₁), or third (τ₂) monomers under a positive (left panels) or a negative (right panels) applied voltage when inserted in membranes composed of DPhPC (a), DPhPS (b), or DPhPC/DOPC (1/1) (c). Solid lines are exponential fittings with one (τ₀) or two (τ₁, τ₂) terms. See Figures S2 and S3 for all distributions displayed individually.