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[摘要]：Transport or 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 Cl, CII, CIII, and ON, the FOF1-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 IC50 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 IC50 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. |
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