The antiport mechanism of the human LAT1 transporter: defining the molecular determinants by means of wet and dry approaches.

LAT1 (SLC7A5) catalyzes an antiport of essential amino acids at the Blood Brain Barrier and placenta barrier where it is mainly expressed. LAT1 becomes overexpressed in human cancers, even when originating from tissues in which it is not highly expressed. The antiport mediated by the transporter is crucial for maintaining the essential amino acid homeostasis in the brain, as demonstrated by the occurrence of Autism Spectrum Disorder forms caused by inherited mutations of LAT1. We have investigated the molecular determinants of the antiport reaction by using wet and dry lab approaches based on proteoliposome transport assay with WT and site-directed mutants coupled with computational analyses. The transport of [H]-histidine, a high-affinity LAT1 substrate, has been assayed on the F400V single and the F400V/K204Q double mutants in comparison with the WT. The F400V mutant showed a strongly impaired antiport function with a concomitant increase of the uniport activity with respect to the WT. The F400V function was scarcely dependent on the pH and was stimulated by cholesterol, similar to previous observations on the WT. The observed impaired stimulation by the countersubstrate was more dramatic in the double mutant that became completely insensitive to the counter-substrate and even trans-inhibited. Molecular dynamics supported the wet lab data showing a much higher degree of mobility of the double mutant, with respect to the WT, indicating the decrease or loss of constraints in the protein conformational changes during the transport cycle.