Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels.
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.
Biomade Technology Foundation, Groningen, The Netherlands. miediema@biomade.nl
Henk
H
Miedema
Anita
A
Meter-Arkema
Jenny
J
Wierenga
John
J
Tang
Bob
B
Eisenberg
Wolfgang
W
Nonner
Hans
H
Hektor
Dirk
D
Gillespie
Wim
W
Meijberg
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002463:Cell Membrane Permeability; D015640:Ion Channel Gating; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D010539:Permeability; D018272:Porins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016062:Porosity; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D020746:Calcium Channels, L-Type; C109237:OmpF protein; D018272:Porins; D011994:Recombinant Proteins; D012964:Sodium; D002118:Calcium