Measuring interference of drug-like molecules with the respiratory chain: toward the early identification of mitochondrial uncouplers in lead finding.

The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I-V proteins in the ETC.

Development of an assay for Complex I/Complex III of the respiratory chain using solid supported membranes and its application in mitochondrial toxicity screening in drug discovery.

Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity.

Electrophysiology of respiratory chain complexes and the ADP-ATP exchanger in native mitochondrial membranes.

Transport of protons and solutes across mitochondrial membranes is essential for many physiological processes. However, neither the proton-pumping respiratory chain complexes nor the mitochondrial secondary active solute transport proteins have been characterized electrophysiologically in their native environment. In this study, solid-supported membrane (SSM) technology was applied for electrical measurements of respiratory chain complexes CI, CII, CIII, and CIV, the F(O)F(1)-ATPase/synthase (CV), and the adenine nucleotide translocase (ANT) in inner membranes of pig heart mitochondria.

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions.

Transporter assays using solid supported membranes: a novel screening platform for drug discovery.

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.