Nigericin and hexylamine effects on localized proton gradients in thylakoids

Abstract

Low concentrations of nigericin and amines such as hexylamine have been suggested to convert chloroplast thylakoid membrane localized proton gradients to a delocalized mode by transferring H + ions into the lumen (De Kouchkovsky, Y. et al. (1986) VII Int. Congr. Photosynth. III, 169). Experiments to test that hypothesis have been done using two assay systems that provide robust indicators of a shift from localized to delocalized energy coupling. Thylakoids stored in low salt medium normally show localized coupling (lumen pH remains considerably more alkaline than the p K a of pyridine (5.44) in both assays and storage in high salt medium yields a delocalized coupling pattern (lumen pH drops to values close to the p K a of pyridine). Therefore, applying those assays to low-salt-stored thylakoids, with and without nigericin or hexylamine, provided the test for whether those compounds caused delocalization of the H + gradient. The assays were: (A) the effect of pyridine (a membrane-permeable buffer) on the ATP formation onset lag in flashing light; (B) permeable buffer (pyridine or hydroxyethylmorpholine) effects on H + uptake into the lumen in steady illumination under coupled compared to basal conditions. By both criteria, it was found that 1–3 nM nigericin caused much more acidification of the lumen compared to the control (absence of nigericin) in low-salt-stored thylakoids. The apparent delocalization of domain H + gradients into the lumen occurred at nigericin concentrations noted by earlier work to cause stimulation of both electron transport and ATP formation (Giersch, G. (1983) Biochim. Biophys. Acta 725, 309–319). In contrast, hexylamine at 50–100 μM did not cause acidification of the lumen, although it clearly had perturbing effects on the membrane domain metastable H + pool. At hexylamine concentrations less than 100 μM there may be transfer of protonated hexylamine into the lumen, although we could not detect it. The high p K a (10.2) of hexylamine would keep the amine in the protonated form and not contribute to lumen acidification.