Molecular mechanism of inhibition of the mitochondrial carnitine/acylcarnitine transporter by omeprazole revealed by proteoliposome assay, mutagenesis and bioinformatics

Abstract

The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC50 of the inhibition of the WT and the C-less CACT by omeprazole were 5.4 µM and 29 µM, respectively. Inhibition kinetics showed non competitive inhibition of the WT and competitive inhibition of the C-less. The presence of carnitine or acylcarnitines during the incubation of the proteoliposomes with omeprazole increased the inhibition. Using site-directed Cys mutants it was demonstrated that C283 and C136 were essential for covalent inhibition. Molecular docking of omeprazole with CACT indicated the formation of both covalent interactions with C136 and C283 and non-covalent interactions in agreement with the experimental data.

Figure 1. Effect of omeprazole on the carnitine antiport mediated by CACT.

Transport was measured as described in Materials and methods adding 0.1[³H]carnitine at time zero to proteoliposomes containing 15 mM carnitine in the absence (○,▾,▴) or in the presence of activated (•, ▪) or non-activated (□) 30 µM omeprazole. In (▾) 2 mM DTE was added 2 min before the transport start. In (▵) 2 mM DTE was added 10 min after the start to the proteoliposomes treated with omeprazole. In (▿) the proteoliposomes were pre-incubated with omeprazole and passed through Sephadex G-75 column to remove the non reacted reagent before the transport assay. The transport reaction was stopped at the indicated times, as described in Materials and methods. Reported values are means ± S.D. from three experiments of mmol of transported substrate per g of protein (mmol · g protein⁻¹).

Figure 2. Dose-response curves for the inhibition of the reconstituted WT and C-less CACT by omeprazole.

The carnitine/carnitine antiport rate was measured as described in Materials and methods, adding 0.1 mM [³H]carnitine together with the indicated concentrations of omeprazole to proteoliposomes reconstituted with WT (•) or C-less (○) CACT. % residual activities with respect to the control are reported. The values are means ± S.D. from three experiments.

Figure 3. Kinetic analysis of the inhibition of the reconstituted CACT by omeprazole.

The carnitine/carnitine antiport rate was measured, as described in Materials and methods, adding [³H]carnitine at different concentrations to proteoliposomes reconstituted with WT CACT (A–C) or C-less CACT (B) containing 15 mM carnitine, in the absence (○) or in the presence of external omeprazole to proteoliposomes. In A, B and C the respective omeprazole concentrations were: 5-15–20 µM (•) and 10–40–68 µM (□). In C, 2 mM DTE was added together with omeprazole. Experimental data plotted according to Lineweaver-Burk as reciprocal transport rate vs reciprocal carnitine concentrations. Reported values are means ± S.D. from three experiments of reciprocal of µmol of transported substrate per mg of protein per min (min · mg · µmol⁻¹).

Figure 4. Effect of substrates on CACT inhibition by omeprazole.

Carnitine (1 mM grey;15 mM white column), octanoyl-carnitine (oct-carn: 0.05 mM grey; 0.5 mM white column) or palmitoyl-carnitine (palm-carn: 0.02 mM grey; 0.1 mM white column) were added together with omeprazole to proteoliposomes reconstituted with the WT CACT and previously passed onto a Sephadex G75 column to remove the external carnitine. After 5 min incubation un-reacted compounds were removed by passing the proteoliposomes through Sephadex G-75 as described in Materials and methods. Transport was started adding 0.1 mM [³H]carnitine and stopped after 30 min. Percent residual activity is reported with respect to the control without the addition of omeprazole. The data represent means ± S.D. of three independent experiments. Significantly different from the sample treated with omeprazole (white column-none) in the absence of substrates, as estimated by the Student's t test (**p<0.01).

Figure 5. Inhibition of WT and Cys mutants of CACT.

Transport rate was measured by adding 0.1 mM [³H]carnitine to proteoliposomes and stopped after 30 min. Omeprazole 30 µM was added to the proteoliposomes reconstituted with the various mutants and incubated for 5 min. The un-reacted compound was removed by passing the proteoliposomes through Sephadex G-75 as described in Materials and methods. The % inhibition with respect to the control are reported. In the legend the Cys residues substituted in the mutant proteins are indicated. The values are means ± S.D. from three experiments. Significantly different from the WT CACT inhibited by omeprazole (WT column), as estimated by the Student's t test (*p<0.05; **p<0.01).

Figure 6. Effect of omeprazole on the carnitine efflux from proteoliposomes.

Efflux of 15[³H]carnitine from pre-labeled proteoliposomes was measured as described in Materials and methods in the presence of only external 10 mM Pipes pH 7.0 (no addition), omeprazole 0.15 mM, 5 mM DTE and carnitine 0.5 mM according to the legend added at time 0. The values are means ± S.D. from three experiments.

Figure 7. Interaction of omeprazole with CACT.

(A) Ribbon representation of CACT with two bound molecules of omeprazole: one covalently bound to C283, as computed through LowModeMD; another non-covalently bound inside the CACT central cavity, as computed through molecular docking on the top-scoring covalent complex previously obtained thorugh LowModeMD. The molecular surface of the protein, close to the ligands, is dotted in yellow. (B) ligand interaction plot representing the CACT residues involved in omeprazole molecular recognition. The depicted complex is the top-scoring one as obtained through molecular docking. Associated docking score and computed binding free energy are reported in Table 1. A caption describing all the detected interactions is included in the picture.