Complete tracking of proton flow in thylakoids - the unit conductance of CFo is greater than 10 fS

We investigated the proton conductance of the channel portion of chloroplast ATP synthase (CFa). Thylakoids were CFi-depleted by EDTA treatment. Proton pumps were stimulated by short flashes of light. Proton flux through CFo was measured spectrophotometrically in three different ways: as proton efflux from the lumen (via neutral red), charge flow across the membrane (via electrochromism) and proton influx into the medium (via phenol red). Hence we completely tracked the protons on their way from the lumen through CF,, into the medium. A first treatment with EDTA removed up to 12% of total CFi without increasing the proton permeability of the membranes. A second treatment removed a further 20% of CFi and increased the proton permeability of membranes by 3 orders of magnitude. The electric potential difference and the pH transients, in both the lumen and medium, decayed with a relaxation time of 7 ms indicating electrically driven proton flow through CFo. If the electric driving force was shunted (e.g. by added gramicidin) both pH transients decayed at 85 ms compared with 2&60 s in control thylakoids. The longer relaxation time under chemical driving force was attributable to larger chemical than electrical capacitance of thylakoids. We calculated a lower limit of the unit conductance of CFO under the assumption that all exposed CFo were proton conducting. The value was 10 fS, corresponding to the passage of 6200 protons/s per CFa (at 100 mV electric driving force) and by orders of magnitude higher than so far reported for any F. channel.