title: The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel.
abstract: Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, TX 77204-5513, USA.
authors.forename: N
authors.initials: N
authors.lastname: Liu
authors.forename: A H
authors.initials: AH
authors.lastname: Delcour
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012492:Salts / Q000737:chemistry*
chemical_list: C113724:OmpC protein; D018272:Porins; D012492:Salts