Calcium gating of proton fluxes in chloroplast thylakoid membranes

Dennis Craig Wooten, Purdue University

Abstract

Acid-base transitions were done to test the prediction that a calcium gating function would block the utilization of lumenal protons derived from lumenally loaded succinic acid. Acid-base transition ATP formation can be driven at least partially by protons derived from succinic acid loaded in the thylakoid lumen. Chlorpromazine, a photoaffinity probe that has high affinity for calcium-induced binding sites in calcium binding proteins, binds covalently to the 8 kDa CF$\sb0$ subunit when the calcium ion concentration is high enough to maintain the localized energy coupling mode. The photoaffinity bound chlorpromazine is suggested to stabilize the "gate closed" configuration. Photoaffinity bound chlorpromazine blocked 50% or more of the succinic acid-dependent acid-base jump ATP formation if the acid stage was near pH 5.5. More acidic conditions resulted in little or no inhibition. Thylakoids that were maintained in the "open gate" configuration, by displacement of calcium with 100 mM KCl, showed no inhibition after photoaffinity treatment with chlorpromazine. However, if CaCl$\sb2$ was added to the KCl medium prior to photoaffinity treatment, chlorpromazine blocked about 50% of the acid-base transition ATP formation. Chlorpromazine is known to enhance Ca$\sp{2+}$ binding in calcium binding proteins such as calmodulin and troponin-C. Chlorpromazine was also shown to block redox-derived protons from entering the lumen at low light intensities, but not at higher light intensities which would allow higher H$\sp+$ accumulation levels. Labeling studies showed that membrane energization (H$\sp+$ uptake) affects the level of ($\sp3$H) chlorpromazine labeling. Additionally, reduction of internal calcium stores by a calcium ionophore (A23187) caused decreased ($\sp3$H) chlorpromazine labeling in deenergized thylakoids. ($\sp3$H) chlorpromazine labeling of isolated CF$\sb0$CF$\sb1$ complex incorporated into azolectin liposomes showed that the 8 kDa CF$\sb0$ subunit III is the only known CF$\sb0$CF$\sb1$ subunit that becomes heavily labeled. A 6 kDa and a 5 kDa band were also heavily labeled. These data lead to the conclusion that Ca$\sp{2+}$ bound to the lumenal side of the CF$\sb0$H$\sp+$ channel can block H$\sp+$ flux from either direction. This is in apparent agreement with the calcium gating hypothesis. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Dilley, Purdue University.

Subject Area

Biochemistry|Biophysics|Biology

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