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

¹ Biomade Technology Foundation
² Rush University Medical Center
³ University of Miami Miller School of Medicine

^* To whom correspondence should be addressed. E-mail: wnonner{at}chroma.med.miami.edu.

Submitted on April 13, 2006
Revised on May 19, 2006
Accepted on 7 September 2006

	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⁺ vs. 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 the modification with the larger glutathione. Zero-current potential measurements revealed a Ca²⁺ vs. 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 DFT/PNP 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⁺.

Key Words: Ca2+ channel, Ca2+ selectivity, MTSES, OmpF, glutathione, pore volume

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