Single channel H+ currents through reconstituted chloroplast ATP synthase CF0-CF1.
The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes, blocked the channel almost completely. Our results, which reveal a high channel unit conductance, and at membrane voltages less than 100 mV low open probability with concomitant mean open times in the micros timescale (less than 100 micros) for the energy coupling in the enzyme complex. At physiological membrane voltages for photophosphorylation (about 30 mV) the enzyme complex would then display a time-averaged conductance of about 1 fS.
Wagner
R
R
Biophysik, Universität Osnabrück, Fachbereich Biologie/Chemie, FRG.
Apley
E C
EC
Hanke
W
W
D002736:Chloroplasts / Q000201:enzymology*; D004553:Electric Conductivity; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons
D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D011522:Protons; D006180:Proton-Translocating ATPases