Permeation Properties of an Engineered Bacterial OmpF Porin Containing the EEEE-Locus of Ca2+ Channels

Abstract

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+. The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of charge/space competition theory.

Crystal Structure of the Monomeric Porin OmpG

“…9 The larger size of the OmpG channel is apparent in liposome-swelling experiments, in which OmpG was found to be much more efficient than OmpC in the transport of various sugars. 10 By contrast, the single-channel conductance of OmpG (∼0.8 nS in 1 M KCl) 8 is substantially smaller than the singlechannel conductance values of OmpF (∼1.4 nS) 13 and OmpC (1.0 nS) 14 measured under similar conditions. Since the OmpG pore is considerably larger than those of OmpC/OmpF (Figure 4(c) and (d)), the ion conductance of OmpG can be regarded as relatively low.…”

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.…”

Ionic Coulomb blockade and anomalous mole fraction effect in the NaChBac bacterial ion channel and its charge-varied mutants

“…2 ) barrier-less conduction, and can be related to the OmpF porin [11,35]. -This state is followed by the Z 1 blocked state, and then by the M 1 state describing double-ion knock-on and identified with L-type calcium channels [10].…”