Il trasportatore di carnitina OCTN2: espressione ed implicazioni fisiopatologiche

Abstract

In the present work we studied the plasma membrane transporter of carnitine OCTN2. We firstly investigated the interaction of this transporter with a commonly used drug, the omeprazolo. The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstituted carnitine transporter catalysed a first-order antiport reaction of carnitine with itself or other substrates stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. Omeprazole externally added to the proteoliposomes, inhibited the carnitine/carnitine antiport catalysed by the reconstituted transporter. The inhibition was reversed by the treatment of the proteoliposomes by DTE indicating that the inhibition was caused by the formation of omeprazole-transporter mixed disulphide. However, omeprazole caused inhibition of the transport also in the presence of DTE, indicating a second inhibition mechanism of non-covalent nature. The presence of the substrate during the incubation of the omeprazole with the proteoliposomes increased the formation of the mixed-disulphide. Omeprazole did not inhibited when present in the internal proteoliposomal compartment, indicating that the inhibition was specifically due to interaction with the external residues or sites of the protein. Omeprazole was not found to be transported by OCTN2: the structure of the activated form of omeprazole, indeed, possess a charged pyridine group and a methoxyl oxygen at a reciprocal distance of three carbon atoms which fulfil the requirement for interaction with the active site and inhibition but not for transport. The implication of the two mechanism of inhibition of the carnitine transporter in physiopathology may be relevant in the light of the omeprazole concentration reached in the blood after administration: the inhibition by omeprazole, indeed, may lead to a carnitine deficiency-like syndrome more or less evident, depending on the dose and on the individual capacity of metabolizing the drug. In the present work we studied, also, the human isoform of OCTN2: (i) we obtained the heterologous over-expression and the purification of hOCTN2 in a bacterial host, E. coli and (ii) we evaluated the expression profile of hOCTN2 in different cancer cell lines and in keratinocytes retrotransduced with HPV16 E6E7. The cDNA coding for hOCTN2 was cloned in two bacterial expression vector and, following two different strategies, we over-expressed the hOCTN2 protein as fusion protein with GST and as hOCTN2 without any tag but with codon bias. In both cases the protein was present in the insoluble fraction of induced lysate and was, then, solubilized with a ionic detergent, Sarkosyl, together with chaotropic agent, Urea. After solubilization the protein was purified on Nichel-chelating chromatography column, obtaining the protein solubilized in Triton X- 100. In the present work we described the expression profile of hOCTN2 in different cancer cell lines and in keratinocytes immortalized by HPV16E6E7: we observed a down-regulation at mRNA levels of hOCTN2 which was reversed by treating cells with 5-aza-cytidine a DNA demetilating agent. Cells over- expressing hOCTN2 were more sensitive to the chemotherapic agent cisplatin. This observation is a preliminary result suggesting a potential role of hOCTN2 in carcinogenesis or in cancer development.