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 (−1
e
) 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
+
.
