Isoflurane selectively inhibits distal mitochondrial complex I in Caenorhabditis elegans.

Abstract

Complex I of the electron transport chain (ETC) is a possible target of volatile anesthetics (VAs). Complex I enzymatic activities are inhibited by VAs, and dysfunction of complex I can lead to hypersensitivity to VAs in worms and in people. Mutant analysis in Caenorhabditis (C.) elegans suggests that VAs may specifically interfere with complex I function at the binding site for its substrate ubiquinone. We hypothesized that isoflurane inhibits electron transport by competing with ubiquinone for binding to complex I. Wildtype and mutant C. elegans were used to study the effects of isoflurane on isolated mitochondria. Enzymatic activities of the ETC were assayed and dose-response curves determined using established techniques. Two-dimensional native gels of mitochondrial proteins were performed after exposure of mitochondria to isoflurane. Complex I is the most sensitive component of the ETC to isoflurane inhibition; however, the proximal portion of complex I (the flavoprotein) is relatively insensitive to isoflurane. Isoflurane and quinone do not compete for a common binding site on complex I. The absolute rate of complex I enzymatic activity in vitro does not predict immobilization of the animal by isoflurane. Isoflurane had no measurable effect on stability of mitochondrial supercomplexes. Reduction of ubiquinone by complex I displayed positive cooperative kinetics not disrupted by isoflurane. Isoflurane directly inhibits complex I at a site distal to the flavoprotein subcomplex. However, we have excluded our original hypothesis that isoflurane and ubiquinone compete for a common hydrophobic binding site on complex I. In addition, immobilization of the nematode by isoflurane is not due to limiting absolute amounts of complex I electron transport as measured in isolated mitochondria.