Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF.
The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.
Baslé
Arnaud
A
Department of Biology and Biochemistry, University of Houston, 369 Science and Research Building 2, Houston, TX 77205-5001, USA.
Qutub
Randa
R
Mehrazin
Mahsa
M
Wibbenmeyer
Jamie
J
Delcour
Anne H
AH
D020816:Amino Acid Motifs / Q000235:genetics; D004594:Electrophysiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D018272:Porins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins