Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase, and, for comparison, gramicidin

Abstract

Summary. We previously introduced a flash spectrophotometricmethod to analyze proton conduction by CF0 in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Sch6n-knecht & W. Junge, 1986, Eur. J. Biochem. 160:627-634). The unit conductance of CF0, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channel G, and the second denoting the average number of active channels per vesicle h-. This technique was applied to CF0, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable ways, i.e. g by addition of known inhibitors of F0-type proton channels and G via the temperature. For gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF0, whose conductance so far has not been accessible to other, more conventional approaches. The timeaveraged unit conductance of CF0 was about 1 pS, equivalent to the turnover of 6 x l0 t H+/(CF0 -sec) at 100 mV driving force.

Further investigation on the high-conductance ion channel of the inner membrane of mitochondria

“…The lack of action of oligomycin and DCCD confirms this interpretation. This conclusion is important also in light of recent reports that some F0 molecules of the chloroplast ATP synthase could have a much higher conductance (in the pS range) than that calculated from the proton current through F 0 inserted into liposomes (Lill et al, 1987;Lill and Junge, 1989).…”

Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation

“…Reports of protein translocation pores in other membrane systems have described single-channel conductivities from 10 pS to 1.5 nS (14)(15)(16)(17)(18)29), and it remained to be established that the carotenoid electrochromic shift could detect pores with similar conductivities in thylakoid membranes. To address this issue, we evaluated the intrinsic sensitivity of the electrochromic measurement by using the pore-forming ionophore gramicidin D. This compound is the most extensively characterized pore to date, and its single-channel conductivity in thylakoids has been determined empirically by using the carotenoid electrochromic shift and confirmed with theoretical calculations (30). From this work, we estimated the time-averaged unit conductance of gramicidin under our assay conditions to be 8.2 pS, corresponding to a flux of approximately 5 ϫ 10 6 ions per s at a 100-mV membrane potential.…”

“…The unit conductance of gramicidin under our assay conditions was calculated as outlined in ref. 30.…”

Inactive Photosystem II Complexes in Leaves

“…The rate of decay of the electric field is due to the movement of unpaired charge across the membrane (29), which is strongly affected by the activation state ofthe ATPase (16). The focus of our experiment is only on the relative amplitudes of the rapid absorbance increases AAFLU and AAFL2, which are easy to determine because the decay of the electrochromic shift is much slower than the rise time ( Fig.…”