Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli.
We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.
Franklin
Michael J
MJ
Department of Physiology and Biochemistry, Wayne State University School of Medicine, Detroit, Michigan, USA.
Brusilow
William S A
WS
Woodbury
Dixon J
DJ
D000465:Algorithms; D004553:Electric Conductivity; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008081:Liposomes / Q000737:chemistry*; D008956:Models, Chemical; D006180:Proton-Translocating ATPases / Q000032:analysis* / Q000737:chemistry*; D011522:Protons
D008081:Liposomes; D011522:Protons; D006180:Proton-Translocating ATPases