Divergent metabolomic signatures by LAT1 and LAT2 amino acid
transporters and characterization of biased LAT1 inhibitors and
mutations
Abstract
Background and Purpose L-type amino acid (AA) transporters LAT1 (SLC7A5)
and LAT2 (SLC7A8) facilitate the bidirectional transport of branched and
aromatic AA across the plasma membrane. While LAT1 is overexpressed in
different tumour cells and it is dedicated to deliver AA into growing
cells, LAT2 facilitates the transcellular AA transport at biological
barriers. Data on dynamic AA transport by LAT1/2 in physiological media
are widely lacking and the impact of LAT1-selective inhibitors and
mutations on the cellular metabolome is unknown. Experimental Approach
The human MDST8 cell line lacking LAT1/2 expression was employed to
generate transiently and stably expressing MDST8-LAT1 and MDST8-LAT2
cells. Together with the HT-29 cell line, we depicted metabolic
signatures mediated by LAT1 and LAT2 using LC-ESI-MS/MS and
characterized potent LAT1/2 inhibitors for their selectivity and mode of
action. Moreover, LAT1 mutations associated with autism spectrum
disorder (ASD) were functionally evaluated. Key Results LAT1 and LAT2
expression induced the expression of 4F2hc and facilitated differential
cellular metabolomic signatures in MDST8 cells. The LAT1(A246V) mutation
showed overall reduced aromatic AA uptake and profound alterations of
intracellular metabolites and biochemical processes. The equipotent LAT1
inhibitors JPH203 and JX-078 showed intriguing differences on
metabolomic effects, uncovering their distinct mode of action.
Conclusion and Implications Our study demonstrates that SLC7A
transporters mediate differential dynamic cellular AA changes under
physiological conditions and reports the broad effects of one human LAT1
mutation associated with ADS. Moreover, the characterization of biased
LAT1 inhibitors challenges current concepts about transporter
pharmacology and has implications for drug discovery.