Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine

Abstract

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

A rapid and simple non-radioactive assay for measuring uptake by solute carrier transporters

“…The existence of these advantages raised the intriguing question of whether using stable isotope labeled substrate as SLC substrate and LC-MS/MS as the detection method could develop an alternative to radioligand uptake assay for SLC. Although several uptake assays using stable isotope labeled substrates have been applied to study SLC functions, none has reported both the optimization of assay and the comparison to the radioactive method ( Gauthier-Coles et al, 2021 ; Bay et al, 2022 ; Papalazarou et al, 2023 ).…”

Impact of Solute Carrier Transporters in Glioma Pathology: A Comprehensive Review

“…Additionally, its expression is correlated with enhanced blood cell formation and metastasis. L-leucine was shown to switch on LAT-1 in cells and enable its uptake due to its high affinity for the transporter [65]. Moreover, a positive association of LAT1 with Ki67 and increased LAT1 expression with worse survival has been demonstrated [62].…”

Uptake Transporters at the Blood–Brain Barrier and Their Role in Brain Drug Disposition

“…Other than FDA-recommended uptake transporters in the human brain, there are other uptake transporters essential for brain physiology [50]; LAT1(SLC 7A5/SLC2A3), which is responsible for the uptake of large neutral amino acids, thyroid hormones, and medicines, is one of them [51,52]. LAT1 is a light-chain amino acid uptake transporter connected by a disulfide bond to the heavy chain 4F2 cell-surface antigen heavy chain (4F2hc).…”