Voltage gating of Escherichia coli porin channels: Role of the constriction loop

Abstract

In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the ␤-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the highresolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C͞A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (⌬116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.Outer membranes of Escherichia coli provide efficient protection from noxious agents in the environment, such as detergents, toxins, and proteolytic enzymes. Their asymmetrical structure, glycolipids in the outer leaflets and phospholipids in the inner, constitutes an impermeable barrier that requires diffusion pathways for the translocation of nutrients and metabolites. In Gram-negative bacteria, this task is accomplished largely by porins. In these trimeric proteins, with masses of 37,000 Da per subunit, each monomer consist of a ␤-barrel that harbors the water-filled transmembrane channel. The pathway is constricted by a loop (L3) which connects ␤-strands 5 and 6 of the barrel and protrudes into the channel lumen, narrowing it midway across the membrane to about 7 ϫ 11 Å (Fig. 1). The passage of solutes across the channel is limited to small (Ͻ600 Da), polar molecules and is slightly cation-selective in OmpF porin (4). Only a few larger solutes require specialized translocation pathways (5). We have studied the structures of this protein to high resolution in both structural and functional terms (2,3,(6)(7)(8)(9).The equilibrium between open and closed states of the channels depends on the applied voltage (7). This phenomenon is poorly understood (10), since the overall potential difference across the outer membrane has been found to be small (11). However, a significant electrostatic field exists parallel to the plane of the membrane in the constriction site (1, 6), and its constituent charges may be affected by an applied potential. This hypothesis is vindicated by the results obtained with replacements of ionizable groups by uncharged residues (2). Also, it has recently been described that polysaccharides (8), membrane-derived oligos...

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…Therefore, the movement of the loop containing the acidic residues in response to voltage is in principle possible, as shown by theoretical analysis (710). However, when the loop was fixed to the barrel wall by a disulfide bond, the gating still occurred (29,194,492), a result that rules out at least a largescale movement of the loop against the barrel wall. Small movements in parts of the loop are still possible, and some molecular dynamics simulation studies (628,659) indeed seem to support this idea.…”

Substitutions in the Eyelet Region Disrupt Cefepime Diffusion through the Escherichia coli OmpF Channel

“…Of significant importance is that the zwitterionic cefepime will presumably form one or several salt bridges during diffusion. Taking into account the modelling of mutations at position 119 and the distribution of charges inside the channel (12,26,33,36), a strong deviation of the bulky cefepime, which is a very large molecule relative to pore diameter (1), probably occurred through the newly orientated electrostatic field. The 132A substitution eliminated the hydrogen bond with Y102 residue and released this position from the positively charged cluster, while the 132D substitution preserved a hydrogen bond with Y102.…”

“…The protein modelling was carried out by Synt:em (Nimes, France). The frame structures used for modelling were wild-type OmpF (6) and the mutants R42C, R82C, G119D, D113G, and deletion 109-114 (11,14,26,29). These structures were analyzed with the COMPOSER program (3) to search the structurally conserved area (24,30).…”

Refolded Outer Membrane Protein A of Escherichia coliForms Ion Channels with Two Conductance States in Planar Lipid Bilayers

“…When incorporated into black lipid membranes, porins exhibit single channel activities that strongly depend on the particular porin, the applied transmembrane potential, and the type and concentration of the electrolyte in the environment. For example, OmpF of Escherichia coli has a conductance of 0.8 nanosiemens in 1 M NaCl, whereas the major porin from Rhodopseudomonas blastica, which is of similar size, exhibits a conductance of 3.9 nanosiemens in 1 M KCl (5,6) OmpA is another major outer membrane protein of E. coli. Although the mass of OmpA is similar to that of many other porins, OmpA consists of two separate domains.…”