The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with the surrounding lipidmicroenvironment to function.Among the main lipid species that constitute eukaryotic cells, cholesterol has highly dynamic levels under a number of physiopathological conditions. This work describes how plasmamembrane cholesterolmodulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterolsaturated methyl-β-cyclodextrin (mβcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and Western blotting. A 20-min preincubation with mβcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison with controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dosedependentmanner. Carnitine uptake in cells incubatedwith empty mβcd and cholesterol-saturated mβcd to preserve the cholesterol content was comparable with controls, suggesting that the mβcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity forNa+ and, in turn, the L-carnitinemaximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipidinduced toxicity and oxidative stress.

Cholesterol stimulates the cellular uptake of L-carnitine by the carnitine/organic cation transporter novel 2 (OCTN2)

Console L.;Scalise M.;Indiveri C.;
2021

Abstract

The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with the surrounding lipidmicroenvironment to function.Among the main lipid species that constitute eukaryotic cells, cholesterol has highly dynamic levels under a number of physiopathological conditions. This work describes how plasmamembrane cholesterolmodulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterolsaturated methyl-β-cyclodextrin (mβcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and Western blotting. A 20-min preincubation with mβcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison with controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dosedependentmanner. Carnitine uptake in cells incubatedwith empty mβcd and cholesterol-saturated mβcd to preserve the cholesterol content was comparable with controls, suggesting that the mβcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity forNa+ and, in turn, the L-carnitinemaximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipidinduced toxicity and oxidative stress.
L-carnitine
cholesterol
membrane lipid
membrane transport
methyl-β-cyclodextrin
proteoliposomes
Biological Transport
Carnitine
Cholesterol
HEK293 Cells
Humans
Proteolipids
Solute Carrier Family 22 Member 5
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11770/324546
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