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

H. Lill*, Gerd Althoff, Wolfgang Junge

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

We previously introduced a flash spectrophotometric method to analyze proton conduction by CF0 in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & 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 vesiclen. 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 wasy, i.e.n 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 time-averaged unit conductance of CF0 was about 1 pS, equivalent to the turnover of 6×105 H+/(CF0·sec) at 100 mV driving force.

Original languageEnglish
Pages (from-to)69-78
Number of pages10
JournalThe Journal of Membrane Biology
Volume98
Issue number1
DOIs
Publication statusPublished - Feb 1987

Keywords

  • ATP synthase
  • CF
  • flash spectrophotometry
  • gramicidin
  • proton conduction
  • unit conductance

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