Electrophysiology of Functional Coupling of Electron Transport Chain Complexes

Abstract

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.