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.
IonGate Biosciences GmbH, Industriepark Hoechst, Frankfurt am Main, Germany.
Natalie
N
Watzke
Kerstin
K
Diekert
Petr
P
Obrdlik
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004553:Electric Conductivity; D004579:Electron Transport; D055724:Electrophysiological Phenomena; D057075:Enzyme Assays; D006868:Hydrolysis; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation
D026901:Membrane Transport Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate