Isoflurane selectively inhibits distal mitochondrial complex I in Caenorhabditis elegans

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Schematic model of potential mechanisms for the inhibition of complex I activity by isoflurane. (A) The L-shaped complex I normally transports electrons from nicotinamide adenine dinucleotide (NADH) to ubiquinone (UQ, shown in its binding site. Complex I activity depends on proper interactions (green ripples) with associated complexes(complexes III and IV) within a supercomplex. (B) If complex III or IV binds isoflurane, changes in conformation (flash) could indirectly inhibit complex I activity (red ripples). (C) If isoflurane disrupts the integrity of the supercomplex, loss of the activating contacts (flash) may lead to indirect inhibition of complex I. For B and C, UQ is omitted for clarity. (D) Direct inhibition of complex I, in which isoflurane and UQ compete for a common hydrophobic binding site. The other members of the supercomplex were omitted for clarity. (E) Direct inhibition by isoflurane at a site distal to the flavoprotein, without preventing UQ binding. The arrows depict the flow of electrons that is measured by the NADH ferricyanide reductase (NFR) assay.

Figure 2

Effect of isoflurane on mitochondrial electron transport. Each point is the mean of ≥4 independent mitochondria preparations from wildtype N2 (●) and mutant gas-1) ([circo]) unless otherwise noted. Error bars represent standard deviations. Lines where presented, were plotted using the parameters from Table 1 for the best exponential fit. (A) Complex I activity of cholate-solubilized mitochondria measured as rotenone-sensitive nicotinamide adenine dinucleotide (NADH):decylubiquinone reductase. (B) Complex I-III activity measured as rotenone-sensitive NADH:cytochrome c reductase. (C) Complex II-III activity measured as antimycin A-sensitive succinate:cytochrome c reductase (Note: Data points for mutant are plotted offset by + 0.2% isoflurane from their actual positions to visually separate the error bars for the datasets.) (D) Wildtype N2 (●) complex III electron transport measured as antimycin A-sensitive decylubiqinol:cytochrome c reductase. (Here, n = 3) (E) Complex IV activity determined as the azide sensitive respiration of intact isolated wildtype N2 mitochondria supplied with external electron donor TMPD\ascorbate under phosphorylating conditions (“state-3”). (F) NFR, activity of the flavoprotein subcomplex of complex I NFR( measured as NADH:ferricyanide reductase for wildtype N2 (●) and gas-1.

Figure 3

Effect of isoflurane on the stability of I:III:IV supercomplexes. BN/hrCN gel stained with Coomassie Blue. Complexes and supercomplexes of the respiratory chain were first separated by blue native gels (BNG) PAGE, then subjected to 0% (panel A) or 19% (panel B) isoflurane respectively, before being re-electrophoresed by hrCNE in the second dimension. A diagonal line is formed if electron transport chain (ETC) complexes do not separate from their original conformation when run in the second dimension in the harsher detergent, maltoside. When interactions between complexes I, III and IV are weakened, increased banding is seen below the diagonal line noted by I:III:IV. There is no difference in banding patterns after exposure to isoflurane, indicating no increase in dissociation of the I:III₂:IV_n supercomplexes.

Figure 4

Inhibition kinetics (A) Initial rate v of complex I activity (rotenone-sensitive nicotinamide adenine dinucleotide (NADH):decylubiquinone reductase) as a function of the concentration of substrate decylubiquinone S measured in the absence (●) and presence ([circo]) of 5.7% isoflurane. Data points are arithmetic means of ≥6 independent mitochondria preparations with error bars for the SD. Solid and broken curves represent nonlinear least squares fit of data to the modified Hill Equation (Equation 1, Table 2); the calculated V_lim for both curves are indicated by horizontal dotted lines. (B) Residuals Plot for complex I activity as a function of decylubiquinone concentration: Closed symbols (● isoflurane absent, [squlf] 5.7% isoflurane) depict the differences between measured data and the fit result for the sigmoidal model where all 3 parameters of the modified Hill equation, V_lim, K and h, were optimized (Table 2 and Fig. 4A). Open symbols ([circo] isoflurane absent, [squlo] 5.7% isoflurane) show the discrepancy between real data and the best fit to hyperbolic (“Michaelis-Menten”) kinetics where h was held fixed to 1 and only V_lim and K were optimized (data not shown).