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

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 (-1e) 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(+).

Energy variational analysis of ions in water and channels: Field theory for primitive models of complex ionic fluids

“…Channel proteins are described by primitive ͑"reduced"͒ models similar to those used to analyze the selectivity of calcium and sodium channels 14,15,[17][18][19]52,[109][110][111][112] and to guide the construction ͑us-ing the techniques of molecular biology͒ of a real calcium channel protein in the laboratory. 50,113 Similar models predicted complex and subtle properties of the RyR channel before experiments were done in Ͼ100 solutions and in seven mutations, some drastic, removing nearly all permanent charge from the "active site" of the channel. 51,77,[114][115][116] This paper is organized into a ͑1͒ biological introduction, ͑2͒ a theoretical introduction, ͑3͒ a long theoretical section with numbered subsections for clarity in navigation, ͑4͒ a computational methods section, ͑5͒ results, and ͑6͒ discussion.…”

“…Ion concentrations inside cells are controlled by biological ͑nano͒ valves called ion channels 120 and controlled and maintained by ion transporters ͑"pumps" 118,128 ͒ as ions move in pores within proteins across otherwise impermeable lipid membranes. It is surprising, but true, that many complex properties of channels selective for calcium or sodium ions can be understood [14][15][16][17][18][19][49][50][51][52]58,60,67,68,77,[109][110][111][112][113][114][115][129][130][131][132][133] using adaptations of the primitive model of ions in bulk solutions [35][36][37][38]90,98,99 fulfilling the early dreams of biophysical chemists, to some extent.…”

“…We introduce the repulsion energy of solid spheres. 14,15,17,19,[49][50][51][52] In this way, the variational calculus extends the primitive model to spatially complex, nonequilibrium time dependent situations, creating a field theory of ionic solutions.…”

Engineering channels: Atomic biology

“…ompG Bayley's laboratory (10) has now developed another channel for technological use, ompG, and learned to keep it open. ompG is a monomer and so has a much less complex structure than ␣HL, and it resembles other porins in which selectivity can be designed to our purpose (11)(12)(13)(14)(15). ompG, however, has natural gating that interferes with its use.…”

“…It would be interesting to see how well these reduced models describe ompG of Chen et al (10). It would be wonderful if Bayley's lab could then build a calciumselective channel as Miedema (11)(12)(13)(14) has and construct a sodium-selective channel as Boda et al (3) suggest should be possible. Of course, such construction will require an understanding of how the cyclodextrin adapter fits into QompG and how adapters confer selectivity, but those are just the kind of questions that an extraordinarily ingenious laboratory like Bayley's are likely to address and answer.…”