Charge Displacements during ATP-Hydrolysis and Synthesis of the Na+-Transporting FoF1-ATPase of Ilyobacter tartaricus

Burzik, Christiane; Kaim, Georg; Dimroth, Peter; Bamberg, Ernst; Fendler, Klaus

doi:10.1016/s0006-3495(03)74632-3

Cited by 17 publications

(16 citation statements)

References 49 publications

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“…Mitochondrial inhibitors, bongkrekic acid (BA), cyclosporin A (CsA) and carboxyatractyloside (CATR), were obtained from Calbiochem (Nottingham, UK). The ADP analogue used in the present study was a cell permeable caged ADP-K þ purchased from Calbiochem as used by Burzik et al (2003). The cytochrome c immunoassay and caspase-8 fluorometric assay kit were obtained from R&D systems (Abingdon, UK).…”

Section: Methodsmentioning

confidence: 99%

The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

2005

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Oxysterols are oxygenated derivatives of cholesterol that may be formed endogenously or absorbed from the diet. Significant amounts of oxysterols have frequently been identified in foods of animal origin, in particular highly processed foods. To date, oxysterols have been shown to possess diverse biological activities; however, recent attention has focused on their potential role in the development of atherosclerosis. Oxysterols have been reported to induce apoptosis in cells of the arterial wall, a primary process in the development of atheroma. The aim of the present study was to identify the role of the mitochondria in the apoptotic pathways induced by the oxysterols 7b-hydroxycholesterol (7b-OH) and cholesterol-5b,6b-epoxide (b-epoxide) in U937 cells. To this end, we investigated the effects of these oxysterols on mitochondrial membrane potential, caspase-8 activity, the mitochondrial permeability transition pore and cytochrome c release. 7b-OH-induced apoptosis was associated with a loss in mitochondrial membrane potential after 2 h, accompanied by cytochrome c release from the mitochondria into the cytosol after 16 h. Pre-treatment with a range of inhibitors of the mitochondrial permeability transition pore protected against 7b-OH-induced cell death. In contrast, b-epoxide induced a slight increase in caspase-8 activity but had no effect on mitochondrial membrane potential or cytochrome c release. The present results confirm that 7b-OH-induced apoptosis occurs via the mitochondrial pathway and highlights differences in the apoptotic pathways induced by 7b-OH and b-epoxide in U937 cells.

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Section: Methodsmentioning

confidence: 99%

The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

2005

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“…The aCNB-caged GABA was used in this study for the first time to investigate GAT1 reactions elicited by GABA concentration jumps generated by a laser flash. There are several examples reported of competitive inhibition of the biological system under investigation by the respective caged compound (39)(40)(41). In particular, aCNB-GABA is known to inhibit ionotropic GABA receptors (42).…”

Section: Biological Properties Of Acnb-caged-gabamentioning

confidence: 99%

Rapid Substrate-Induced Charge Movements of the GABA Transporter GAT1

Bicho

Grewer

2005

Biophysical Journal

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The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 micros. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic "sodium-occlusion" reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl- may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others.

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“…Liposomes in which the transporter has been incorporated (proteoliposomes) are adsorbed to the SSM, and the currents generated by the transporter activity are recorded via capacitive coupling. [4][5][6][7][8] The compound membrane formed by the adsorbed proteoliposomes and the SSM can withstand high flow velocities, allowing for a fast solution exchange at the surface. By rapidly changing from a solution containing no substrate for the transporter to one that contains a substrate, the transporter can be activated, and a transient current can be recorded that contains information about the size and the temporal development of the transport process.…”

Section: Introductionmentioning

confidence: 99%

Transporter Assays Using Solid Supported Membranes: A Novel Screening Platform for Drug Discovery

Kelety¹,

Diekert²,

Tobien³

et al. 2006

ASSAY and Drug Development Technologies

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Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H(+)-dependent peptide transporter PepT1, the gastric proton pump, and the Na(+)/Ca(2+) exchanger. This technology promises to have an important impact on the drug discovery process.

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Charge Displacements during ATP-Hydrolysis and Synthesis of the Na+-Transporting FoF1-ATPase of Ilyobacter tartaricus

Cited by 17 publications

References 49 publications

The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Rapid Substrate-Induced Charge Movements of the GABA Transporter GAT1

Transporter Assays Using Solid Supported Membranes: A Novel Screening Platform for Drug Discovery

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