Impact of 7-ketocholesterol on the function and stability of the LAT1 transporter

The L-type amino acid transporter (LAT1, SLC7A5) is involved in supplying essential amino acids for brain and fetal development. The regulation of LAT1 function and stability by cholesterol was previously described in ex vivo and in vitro experimental models. The complexity of the relationship between LAT1 and cholesterol has been further studied by investigating the impact of selected oxidized forms of cholesterol, focusing on the most abundant auto-oxidized form of cholesterol, namely 7-ketocholesterol, which increases with age and pathologies characterized by high reactive oxygen species (ROS) levels. The partial substitution of cholesterol with 7-ketocholesterol in HEK293 cells transiently overexpressing the human LAT1 (HEK293-LAT1), decreases the Vmax of LAT1 mediated transport and the thermal stability of the protein. The impairing effect on transport was directly tested in LAT1 reconstituted proteoliposomes supplemented with 7-ketocholesterol. In this experimental model, it was found that the presence of 7-ketocholesterol altered the transport stimulation by adenosine triphosphate (ATP), as previously described. This was likely due to the higher rigidity of 7-ketocholesterol compared to cholesterol, which alters the membrane/protein interaction, weakening the ATP binding to LAT1. LAT1 transport function was also inhibited when proteoliposomes were supplemented with 7α-hydroxycholesterol or 25-hydroxycholesterol. These deleterious effects may be relevant in chronic pathological conditions characterized by altered cholesterol/oxysterol ratios, ranging from metabolic syndrome to kidney disease and neurological disorders.