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System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na+-independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 - 1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.

Les acides aminés ont de nombreuses fonctions dans l’organisme en plus de leur rôle comme constituants élémentaires des protéines. Ils peuvent par exemple servir de neurotransmetteur ou de signal pour l’activation de voies de signalisation intracellulaires. Leur passage à travers la membrane plasmique est facilité par des transporteurs de la famille des protéines SLC. Les transporteurs hétérodimériques d’acides aminés HAT appartiennent aux SLC. Les HAT sont constitués d’une chaîne légère SLC7 assurant la spécificité de transport et d’une chaîne lourde SLC3 impliquée dans l’adressage du complexe protéique à la membrane. Ma thèse a porté sur l’étude du rôle d’un homologue de SLC7 chez la drosophile, Minidiscs (Mnd), dans le fonctionnement du système nerveux. Mnd appartiendrait aux transporteurs du système L, principalement connus pour leur rôle dans la prolifération cellulaire. Mes travaux de thèse ont permis de mettre en évidence la localisation de Mnd dans le cerveau de drosophile dans certains neurones (corps pédonculés, neurones dopaminergiques) et dans certaines cellules gliales (glie corticale). La présence de Mnd dans le cerveau semble intervenir dans la modulation de certains comportements, tels que le réflexe de géotaxie négative. Ces travaux ont aussi montré que, comme les HAT de mammifères, Mnd s’associe de façon covalente à un partenaire protéique. Les expériences de transport semblent par ailleurs confirmer l’appartenance de Mnd au système L.Ces résultats suggèrent que Mnd est probablement impliqué dans la régulation de l’activité neuronale et donc dans le fonctionnement du système nerveux, ce qui n’avait encore jamais été décrit pour un transporteur du système L. / Amino acids have many functions in the body in addition to their role as basic constituents of proteins. They can for example serve as a neurotransmitter or signal for the activation of intracellular pathways. Carriers of the SLC protein family facilitate their path through the plasma membrane. The heterodimeric amino acid transporters HAT belong to SLC proteins. HAT are composed of a light chain SLC7 ensuring the specificity of transport and a heavy chain SLC3 involved in the addressing of the protein complex to the plasma membrane. My thesis focused on studying the role of a SLC7 homologue in drosophila, Minidiscs (Mnd), in the functioning of the nervous system. Mnd might belong to system L carriers, mainly known for their role in cell proliferation. My thesis work led to highlight the location of Mnd in the drosophila brain in some neurons (mushroom bodies, dopaminergic neurons) and some glial cells (cortical glia). The presence of Mnd in the brain seems to be involved in the modulation of some behaviors such as negative geotaxis reflex. This work also showed that, as for mammal HAT, Mnd is associated covalently to a protein partner. Transport experiments seem also to confirm the belonging of Mnd to the system L. These results suggest that Mnd is probably involved in the regulation of neuronal activity and thus in the functioning of the nervous system, which had never been described for a system L carrier.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.