title: Ca2+ selectivity of a chemically modified OmpF with reduced pore volume.
abstract: We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca(2+) channels, and an accessible and reactive cysteine. After chemical modification with the cysteine-specific, negatively charged (-1e) reagents MTSES or glutathione, this LECE mutant was tested for Ca(2+) versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca(2+) mole fractions <10(-3). MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca(2+) chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca(2+) versus monovalent cation selectivity that was highest in the presence of Li(+) and lowest in the presence of Cs(+). Our data clearly show that after the binding of Ca(2+) the LECE pore (even with the bulky glutathione present) is spacious enough to allow monovalent cations to pass. Theoretical computations based on density functional theory combined with Poisson-Nernst-Planck theory and a reduced pore model suggest a functional separation of ionic pathways in the pore, one that is specific for small and highly charged ions, and one that accepts preferentially large ions, such as Cs(+).
authors.affiliation: Biomade Technology Foundation, Nijenborgh, Groningen, The Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: Miedema
authors.forename: Maarten
authors.initials: M
authors.lastname: Vrouenraets
authors.forename: Jenny
authors.initials: J
authors.lastname: Wierenga
authors.forename: Dirk
authors.initials: D
authors.lastname: Gillespie
authors.forename: Bob
authors.initials: B
authors.lastname: Eisenberg
authors.forename: Wim
authors.initials: W
authors.lastname: Meijberg
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Nonner
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002414:Cations, Monovalent / Q000737:chemistry; D002586:Cesium / Q000737:chemistry; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D004553:Electric Conductivity; D004560:Electricity; D005978:Glutathione / Q000737:chemistry; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry; D008094:Lithium / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry
chemical_list: D002414:Cations, Monovalent; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D002586:Cesium; D008094:Lithium; D012964:Sodium; D005978:Glutathione; D003545:Cysteine; D002118:Calcium