Kerstin Diekert

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. Specific substrates and inhibitors were used to validate the different assays, and the corresponding K(0.5) and IC(50) values were in good agreement with previously published results obtained with other methods. In combined measurements of CI-CV, it was possible to detect oxidative phosphorylation (OXPHOS), to measure differential effects of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the respective protein activities, and to determine the corresponding IC(50) values. Moreover, the measurements revealed a tight functional coupling of CI and CIII. Coenzyme Q (CoQ) analogues decylubiquinone (DBQ) and idebenone (Ide) stimulated the CII- and CIII-specific electrical currents but had inverse effects on CI-CIII activity. In summary, the results describe the electrophysiological and pharmacological properties of respiratory chain complexes, OXPHOS, and ANT in native mitochondrial membranes and demonstrate that SSM-based electrophysiology provides new insights into a complex molecular mechanism of the respiratory chain and the associated transport proteins. Besides, the SSM-based approach is suited for highly sensitive and specific testing of diverse respiratory chain modulators such as inhibitors, CoQ analogues, and uncoupling agents.

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. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts.

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. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10 μM) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade.

Electrophysiology of Functional Coupling of Electron Transport Chain Complexes

Electron transport chain in the inner mitochondrial membranes consists of four multisubunit protein complexes CI to CIV which are coupled via electron carrier molecules as well as protein-protein interactions. The coupling of the complexes is essential for the proper functioning of the chain and may be an important factor for regulation and balancing of respiration, ATP synthesis and production of reactive oxygen species. However, direct functional studies on the action of the respiratory chain in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed electrophysiological analyses of electron transport chain in native inner mitochondrial membranes using the solid-supported membranes (SSM) and the SURFE2R technology. The inner mitochondrial membranes were purified from pig heart mitochondria by sucrose gradient fractionation and adsorbed onto SSM sensors. The chain complexes were activated either by NADH for the studies of CI-CIII-CIV coupling, or by succinate and cytochrome c for the analysis of the CII-CIII function. The tested proteins were pharmacologically characterized using specific substrates and inhibitors. Serial application of different inhibitors as well as the coenzyme Q analogs decylubiquinone revealed a tight functional interplay between the complexes CI, CIII, and functional coupling to the complex CIV. The complexes CII and CIII were also functionally coupled. An excess of the coenzyme Q analog idebenone had stimulating effect on the CII-CIII activity but was reducing the CI-CIII-CIV-specific currents. In summary, the presented results demonstrate an easy and reliable approach for studying the complex functional interplay of mitochondrial transport proteins in their native environment, and can help to understand the physiology of different mitochondrial functions. Since different assay conditions can be tested on the same sensor, the technology allows highly effective comparative analysis of the different complex activities.

Cell-Free Electrophysiology of Native Inner Mitochondrial Membranes

Transporters, pumps and channels of inner mitochondrial membranes are involved in all central mitochondrial functions such as regulation of ATP synthesis, thermogenesis, control of cellular calcium, regulation of apoptosis, and in balancing production of cellular reactive oxygen species. However, functional investigation of mitochondrial transport proteins is tedious. Typically, the functional studies rely on the reconstitution of recombinant proteins in proteoliposomes. This work describes the use of the SURFE2R technology for electrical measurements of transporters in native mitochondrial membranes. The inner mitochondrial membranes were isolated from pig heart and adsorbed on gold electrodes according to standard protocols. These biosensors were measured with the standard SURFE2R equipment. Three transport proteins were characterized: the ADP/ATP exchanger (ANT), the F-type ATPase (complex V), and the Cytochrome c Oxidase (COX, complex IV). The transport was activated through substrate concentration jumps (ATP or ADP in case of ANT and F-type ATPase) or, in case of COX, via concentration jumps with reduced cytochrome c. In case of activation via ATP-concentration jumps, both, the ANT and F-type ATPase activity could be detected independently on the same sensor by applying ANT- or ATPase-selective solutions. The specificity of the currents was verified by ATPase- and ANT-specific inhibitors. As expected, the ATP-induced activity of ANT was increased in presence of ADP gradients across the membranes. The ANT-signals depended on the applied ATP-concentration. The cytochrome c-induced currents were inhibited by COX-specific inhibitor cyanide and depended on the redox state of the protein. In summary, the presented results demonstrate a novel, easy and reliable approach for functional studies of mitochondrial transporters and pumps in their native surroundings, and can potentially help to understand the complex molecular mechanisms of different mitochondrial functions. Additionally, the technology platform can also be used to perform pharmacological screens for mitochondria-targeted drugs.