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Ca²⁺ Selectivity of a Chemically Modified OmpF with Reduced Pore Volume

Henk Miedema ^*, Maarten Vrouenraets ^*, Jenny Wierenga ^*, Dirk Gillespie , Bob Eisenberg , Wim Meijberg ^* and Wolfgang Nonner 

^* Biomade Technology Foundation, Nijenborgh, Groningen, The Netherlands; Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois; and Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, Florida

Correspondence: Address reprint requests to W. Nonner, Dept. of Physiology and Biophysics, University of Miami, Miller School of Medicine, PO Box 016430, Miami, FL 33101-6430. E-mail: wnonner{at}chroma.med.miami.edu.

We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca²⁺ 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 Ca²⁺ versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca²⁺ mole fractions <10^–3. MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca²⁺ chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca²⁺ 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 Ca²⁺ 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⁺.

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