Determination of Proton Flux and conductance at pH 6.8 through single F_o sectors from Escherichia coli

¹ Wayne State University
² Brigham Young University

^* To whom correspondence should be addressed. E-mail: dixon_woodbury{at}byu.edu.

Submitted on April 30, 2004
Revised on May 24, 2004
Accepted on 18 August 2004

	Abstract

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H⁺) flux and conductance through a single F_o of the F₁F_o ATP synthase. Lipid vesicles reconstituted with just a few functional F_o 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 CCCP, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single F_o sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wildtype F_o has a proton flux of 3100 ± 500 H⁺/sec/ F_o at a transmembrane potential of 106 mV 25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.

Key Words: F1Fo ATPase, H+ channel, e coli, proton channel, proton coductance, proton transport