Ca²⁺ selectivity of a chemically modified OmpF with reduced pore volume

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 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 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⁺.