Cooperation of Basolateral Amino Acid Transporters in Mouse and Cell Culture Models

Abstract

Amino acids have central roles in every living organism as structural units for proteins, metabolic precursors, osmolytes and signaling molecules. In some heterotrophic organisms, such as humans and mice, some amino acids cannot be synthesized de novo through other metabolic pathways and need to be constantly provided from the diet. This important evolutionary constraint led to the important classification of the 20 known proteinogenic amino acids into nutritionally essential and non-essential ones. In order to achieve all their biological functions, amino acids need to cross cell membranes and because of their chemical partial charges they cannot freely diffuse across the lipid bilayer. To overcome this hurdle, cell membranes are endowed with transmembrane carrier proteins called amino acid transporters. In (re)absorptive epithelia, cells are in a differentiated state with an apical membrane, which is facing an organ-specific lumen, and a basolateral membrane, which is in contact with the extracellular matrix and the blood vessels. In contrast to the apical transport, the mechanism of amino acid transport across the basolateral membrane still remains elusive. The best characterized basolateral amino acid transporters in the small intestine and kidney, i.e. y+LAT1-4F2hc (Slc7a7-Slc3a2) and LAT2-4F2hc (Slc7a8-Slc3a2), are obligatory antiporters and therefore cannot mediate alone the net vectorial amino acid efflux. In order to achieve this task, additional amino acid transporters capable of transporting amino acids unidirectionally, such as the uniporters TAT1 (Slc16a10) and/or LAT4 (Slc43a2), need to be present. TAT1 transports aromatic amino acids and has been recently shown to be important in the regulation of amino acid homestasis in vivo. On the other hand, LAT4 has been shown to transport branched-chain amino acids, phenylalanine and methionine, but information about its potential role in vivo is still missing. Therefore, with the present work we aimed at providing new information about the physiological role of this transporter by characterizing a newly established constitutive Slc43a2-/- knock-out mouse. The Slc43a2-/- mice were born at the expected Mendelian ratio and without any phenotypic difference compared with wild type littermates but showed an early severe phenotype with malnutrition, altered glucose homeostasis, altered hepatic gene expression and premature death. Moreover, by using these Lat4-deficient mice as negative control, we gathered new information about the localization of Lat4 in kidney, small intestine, liver and skeletal muscle. Specifically, we observed a major expression of Lat4 in the basolateral membrane of kidney proximal tubule, thick ascending limb and a minor expression in the distal convoluted tubule. In the small intestine, Lat4 expression was confined to the basolateral membrane of enterocytes with no expression in the intestinal crypts. On the other hand, skeletal muscle and liver did not show any Lat4 expression. In addition to the aforementioned work in vivo, we aimed at deepening the understanding of LAT4 regulation and cooperation in the MDCK cell model. We therefore reconstituted the essential machinery for neutral amino acid transport and validated its function with uptake of radiolabeled amino acids. Moreover, using the Xenopus laevis oocytes expression system we addressed the effect of a predicted serine phosphorylation site on LAT4 function. In both in vitro systems, we provided preliminary information indicating that this phosphorylation downregulates LAT4 function by cellular mechanisms that still need to be investigated in more details. In conclusions, the major achievements of the present thesis consist in the first characterization of a newly generated constitutive Slc43a2-/-knock-out mouse with which we demonstrated that Lat4 is essential for early postnatal development. Moreover, findings obtained using the MDCK cell model and the Xenopus laevis oocytes expression system provided new preliminary information about a potential functional regulation of LAT4 by phosphorylation.