Biophysical Journal
Volume 91, Issue 12, 15 December 2006, Pages 4392-4400
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Ca2+ Selectivity of a Chemically Modified OmpF with Reduced Pore Volume

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Abstract

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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