Quantitative modelling of amino acid transport and homeostasis in mammalian cells

Abstract

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell’s endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.

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

“…In the IC 50 determinations on hCMEC/D3 cells, the baseline uptake of [ 13 C 6 , 15 N]-L-leucine was considerably lower in the presence of BCH compared to JPH203. This is in accordance with the non-selective inhibitory effect of BCH for the LAT system [ 30 , 42 , 47 , 48 ] and other transporters such as B 0 AT2 [ 10 ], which may contribute to baseline uptake of [ 13 C 6 , 15 N]-L-leucine. Because the LAT-1 specific inhibitor JPH203 inhibits the majority of [ 13 C 6 , 15 N]-L-leucine uptake in hCMEC/D3 cells, it can be concluded that LAT-1 HD is the primary transporter responsible for L-leucine uptake in these brain capillary endothelial cells.…”

“…The first, JPH203, is currently studied in a clinical trial and shows high specificity for LAT-1 HD [ 24 , 31 ]. The second, BCH, is a non-selective inhibitor for L-system amino acid transporters and a substrate of LAT-1 [ 30 ], LAT-2 [ 42 ], LAT-3 [ 47 ], and LAT-4 [ 48 ], as well as other amino acid transporters such as B 0 AT2 [ 10 ]. Based on previously reported IC 50 values [ 30 , 31 , 32 , 44 ], the investigated concentration ranges for the two inhibitors were selected.…”

“…As a consequence, LAT-1 HD is dependent on the uptake of amino acids by other amino acid transporters. The information on the complex interplay of amino acid transporters has been summarized elsewhere [ 10 ].…”

“…Direct measurement of amino acid uptake can especially be realized in heterologous expression systems with low intracellular levels of amino acids, such as Xenopus laevis oocytes [ 28 ]. Although 13 C-isotopologes of amino acids have been used for uptake experiments [ 10 ], especially in the context of metabolism studies [ 29 ], previous investigations of L-leucine uptake in the context of transporter characterization were primarily based on the radioactive isotopologes [ 14 C]-L-leucine or [ 3 H]-L-leucine [ 5 , 10 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Nevertheless, through comprehensive uptake studies with different amino acids and the consideration of the respective metabolism, the specific contribution of LAT-1 HD for L-leucine uptake and, hence, functional characterization can be performed [ 10 ].…”

Discovery and synthesis of hydroxy-L-proline based blockers of the neutral amino acid transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2)

“…Assuming poor blood-barrier permeability, the overlap with glutamate transporters described in this work may not be as detrimental in peripheral tissues where glutamate transporters are expressed at a much lower density than within the CNS. 46 Other transporters that should be considered for inhibitor cross reactivity include the neutral amino acid transporters from the SLC38A and SLC7A+SLC3A2 gene families.…”