Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca-channels

¹ Biomade Technology Foundation, Groningen, The Netherlands
² Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago
³ Department of Physiology and Biophysics, University of Miami School of Medicine

^* To whom correspondence should be addressed. E-mail: miedema{at}biomade.nl.

Submitted on February 11, 2004
Revised on March 9, 2004
Accepted on 13 August 2004

	Abstract

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1e 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 non-selective 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 E113 and D117. 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 (CSC) model, introduced by Nonner et al., (2000) and initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume to be key determinants of ion selectivity and 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 an entire different ion channel family, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of CSC theory.

Key Words: CSC, Calcium selectivity, Charge/Space Competition, Ion selectivity, Selectivity filter volume

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