Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

L-type amino acid transporter 1 as a target for inflammatory disease and cancer immunotherapy.

Ingestion of amino acids is fundamental for cellular activity. Amino acids are important components for protein synthesis but are also crucial for intracellular metabolic reactions and signal transduction. Following activation, immune cells induce metabolic reprogramming to generate adequate energy and constitutive substances. Hence, the delivery of amino acids by transporters is necessary for the progression of metabolic rewiring. In this review, we discuss how amino acids and their transporters regulate immune cell functions, with emphasis on LAT1, a transporter of large neutral amino acids. Furthermore, we explore the possibility of targeting amino acid transporters to improve immune disorders and cancer immune therapies.

[Drug Discovery Targeting an Amino Acid Transporter for Diagnosis and Therapy].

Nutrients are essential for all living organisms. Because growing cancer cells have strong metabolic demands, nutrient transporters are constitutively increased to facilitate the nutrient uptake. Among these nutrient transporters, L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids including essential amino acids, is critical for cancer growth. Therefore, LAT1 has been considered as an attractive target for diagnosis and therapy of cancers. We have developed several lines of compounds for cancer diagnosis and therapy. To diagnose cancer by using positron emission tomography (PET) probes, we have created amino acid derivatives which are selectively transported by LAT1 and accumulated in cancer cells. In addition to amino acid derivatives as the LAT1 inhibitors, we also have made non-amino acid small compounds as anti-cancer drugs which inhibit LAT1 function and suppress tumor growth. The LAT1 targeting anti-cancer drug showed low toxicity but strong effects on various types of cancer cells in animal models. The novel PET probe is approved for clinical research and the new anti-cancer drug has been under clinical trial. Small compounds targeting the amino acid transporter bring us new tools for cancer diagnosis and therapy.

L-type amino acid transporter 1 is associated with chemoresistance in breast cancer via the promotion of amino acid metabolism.

18F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.

[Transfer Mechanisms of Compounds between Mother and Fetus/Infant Aimed for Optimized Medication during Pregnancy and Breastfeeding].

Potential risks to the fetus or infant should be considered prior to medication during pregnancy and lactation. It is essential to evaluate the exposure levels of drugs and their related factors in addition to toxicological effects. Epilepsy is one of the most common neurological complications in pregnancy; some women continue to use antiepileptic drugs (AEDs) to control seizures. Benzodiazepines (BZDs) are widely prescribed for several women who experience symptoms such as anxiety and insomnia during the postpartum period. In this review, we describe the 1) transport mechanisms of AEDs across the placenta and the effects of these drugs on placental transporters, and 2) the transfer of BZDs into breast milk. Our findings indicated that carrier systems were involved in the uptake of gabapentin (GBP) and lamotrigine (LTG) in placental trophoblast cell lines. SLC7A5 was the main contributor to GBP transport in placental cells. LTG was transported by a carrier that was sensitive to chloroquine, imipramine, quinidine, and verapamil. Short-term exposure to 16 AEDs had no effect on folic acid uptake in placental cells. However, long-term exposure to valproic acid (VPA) affected the expression of folate carriers (FOLR1, SLC46A1). Furthermore, VPA administration changed the expression levels of various transporters in rat placenta, suggesting that sensitivity to VPA differed across gestational stages. Lastly, we developed a method for quantifying eight BZDs in human breast milk and plasma using LC/MS/MS, and successfully applied it to quantify alprazolam in breast milk and plasma donated by a lactating woman.

Neural production of kynurenic acid in Caenorhabditis elegans requires the AAT-1 transporter.

Kynurenic acid (KynA) levels link peripheral metabolic status to neural functions including learning and memory. Since neural KynA levels dampen learning capacity, KynA reduction has been proposed as a therapeutic strategy for conditions of cognitive deficit such as neurodegeneration. While KynA is generated locally within the nervous system, its precursor, kynurenine (Kyn), is largely derived from peripheral resources. The mechanisms that import Kyn into the nervous system are poorly understood. Here, we provide genetic, anatomical, biochemical, and behavioral evidence showing that in C. elegans an ortholog of the human LAT1 transporter, AAT-1, imports Kyn into sites of KynA production.

JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function.

JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.

Slc7a5 alters Kvß-mediated regulation of Kv1.2.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in neuronal excitability and is regulated by a variety of known and unknown extrinsic factors. The canonical accessory subunit of Kv1.2, Kvß, promotes N-type inactivation and cell surface expression of the channel. We recently reported that a neutral amino acid transporter, Slc7a5, alters the function and expression of Kv1.2. In the current study, we investigated the effects of Slc7a5 on Kv1.2 in the presence of Kvß1.2 subunits. We observed that Slc7a5-induced suppression of Kv1.2 current and protein expression was attenuated with cotransfection of Kvß1.2. However, gating effects mediated by Slc7a5, including disinhibition and a hyperpolarizing shift in channel activation, were observed together with Kvß-mediated inactivation, indicating convergent regulation of Kv1.2 by both regulatory proteins. Slc7a5 influenced several properties of Kvß-induced inactivation of Kv1.2, including accelerated inactivation, a hyperpolarizing shift and greater extent of steady-state inactivation, and delayed recovery from inactivation. These modified inactivation properties were also apparent in altered deactivation of the Kv1.2/Kvß/Slc7a5 channel complex. Taken together, these findings illustrate a functional interaction arising from simultaneous regulation of Kv1.2 by Kvß and Slc7a5, leading to powerful effects on Kv1.2 expression, gating, and overall channel function.

Regulation of Melanogenesis by the Amino Acid Transporter SLC7A5.

Integration of chromatin immunoprecipitation-sequencing and microarray data enabled us to identify previously unreported MITF-target genes, among which the amino acid transporter SLC7A5 is also included. We reported that small interfering RNA-mediated SLC7A5 knockdown decreased pigmentation in B16F10 cells but neither affected morphology nor dendricity. Treatment with the SLC7A5 inhibitors 2-amino-2-norbornanecarboxylic acid (BCH) or JPH203 also decreased melanin synthesis in B16F10 cells. Our findings indicated that BCH was as potent as reference depigmenting agent, kojic acid, but acted through a different pathway not affecting tyrosinase activity. BCH also decreased pigmentation in human MNT1 melanoma cells or normal human melanocytes. Finally, we tested BCH on a more physiological model, using reconstructed human epidermis and confirmed a strong inhibition of pigmentation, revealing the clinical potential of SLC7A5 inhibition and positioning BCH as a depigmenting agent suitable for cosmetic or dermatological intervention in hyperpigmentation diseases.

Alzheimer's Disease Phenotype or Inflammatory Insult Does Not Alter Function of L-Type Amino Acid Transporter 1 in Mouse Blood-Brain Barrier and Primary Astrocytes.

PURPOSE: The study aim was to evaluate the effect of Alzheimer's disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes. METHODS: The Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion. RESULTS: There were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups. CONCLUSIONS: The results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.

High throughput silencing identifies novel genes in endometrioid endometrial cancer.

OBJECTIVE: To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. MATERIALS AND METHODS: A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. RESULTS: Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). CONCLUSION: We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

KEY MESSAGE: Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.

3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 µM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Aspartate-modified doxorubicin on its N-terminal increases drug accumulation in LAT1-overexpressing tumors.

L-type amino acid transporter 1 (LAT1), overexpressed on the membrane of various tumor cells, is a potential target for tumor-targeting therapy. This study aimed to develop a LAT1-mediated chemotherapeutic agent. We screened doxorubicin modified by seven different large neutral amino acids. The aspartate-modified doxorubicin (Asp-DOX) showed the highest affinity (Km = 41.423 µmol/L) to LAT1. Aspartate was attached to the N-terminal of DOX by the amide bond with a free carboxyl and a free amino group on the α-carbon atom of the Asp residue. The product Asp-DOX was characterized by HPLC/MS. In vitro, Asp-DOX exerted stronger inhibition on the cancer cells overexpressing LAT1 and the uptake of Asp-DOX was approximately 3.5-fold higher than that of DOX in HepG2 cells. Pharmacokinetic data also showed that Asp-DOX was expressed over a longer circulation time (t1/2 = 49.14 min) in the blood compared to DOX alone (t1/2 = 15.12 min). In HepG2 and HCT116 tumor-bearing mice, Asp-DOX achieved 3.1-fold and 6.4-fold accumulation of drugs in tumor tissue, respectively, than those of the unmodified DOX. More importantly, treatment of tumor-bearing mice with Asp-DOX showed a significantly stronger inhibition of tumor growth than mice treated with free DOX in HepG2 tumor models. Furthermore, after Asp modification, Asp-DOX avoided MDR mediated by P-glycoprotein. These results suggested that the Asp-DOX modified drug may provide a new treatment strategy for tumors that overexpress LAT1 and MDR1.

The catalytic subunit of the system L1 amino acid transporter (slc7a5) facilitates nutrient signalling in mouse skeletal muscle.

The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass.

LAT1 is a central transporter of essential amino acids in human umbilical vein endothelial cells.

Endothelial cell proliferation supporting angiogenesis requires sufficient nutrient supply because of facilitated intracellular metabolism. However, little is known about the mechanism for the promotion of nutrient incorporation in proliferating endothelial cells. Here we show that L-type amino acid transporter 1 (LAT1) is a major transporter of essential amino acids in human umbilical vein endothelial cells (HUVECs). Growing HUVECs express a certain level of LAT1. A LAT1-specific inhibitor suppressed leucine uptake, cell proliferation, and tube formation of HUVECs. Therefore, LAT1 acts to support effective uptake of amino acids, which is critical for the optimal function of HUVECs for angiogenesis.

LAT-1 functions as a promotor in gastric cancer associated with clinicopathologic features.

L-type amino-acid transporter 1 (LAT-1) is a member of system L-type transporters, essential for cells maintenance and proliferation. However, the role of LAT-1 remains illegible in gastric cancer (GC). In this study, we found that LAT-1 was aberrantly up-regulated in both GC cell lines (MKN-45, MGC-803 and CRL-5974) and human GC specimens. The expression characteristic of LAT-1 in GC was significantly associated with clinicopathologic features such as tumor size, lymph node metastasis, local invasion and TNM stage. By suppressing the expression of LAT-1 in MKN-45 cells, the cell cycle was arrested in G0/G1 phase, and the ability of cell proliferation was significantly decreased in vitro. Moreover, the cell migration and invasion of MKN-45 cells was significantly impaired by knocking down LAT-1. Thus, our results suggest that LAT-1 may function as an oncogene in GC, which provides us a new biomarker in GC and perhaps a potential target for GC prevention, diagnose and therapeutic treatment.

Transport of gabapentin by LAT1 (SLC7A5).

Gabapentin is used in the treatment of epilepsy and neuropathic pain. Gabapentin has high and saturable permeability across the BBB, but no mechanistic studies underpinning this process have been reported. The aim of the current study was to investigate the transport of gabapentin in a model of the BBB, identify the important drug transporter(s) and to use mathematical modelling to quantify the processes involved. A human brain endothelial cell line (hCMEC/D3) was utilised as an in-vitro model of the BBB. Uptake of radiolabeled gabapentin into cells in the presence of chemical inhibitors, siRNA or overexpressed drug transporters of interest was investigated. Gabapentin was demonstrated to be a LAT1 substrate in brain endothelial cells (LAT1-process; Km=530µM and Vmax=7039pmoles/million cells/min versus other-processes; Km=923µM and Vmax=3656pmoles/million cells/min) and in transfected HEK 293 LAT1 cells (LAT1-process; Km=217µM and Vmax=5192pmoles/million cells/min versus otherprocesses; Km=1546µM and Vmax=3375pmoles/million cells/min). At physiological concentrations of gabapentin, LAT1 mediated transport was 3 or ~10-fold higher than the other transport processes in the two systems, respectively, demonstrating clear selectivity for gabapentin. In-silico structural homology modelling confirmed that LAT1 could have the LeuT conserved fold and functions by the alternative access mechanism. Mathematical modelling of this mechanism revealed revised significance of Vmax and Km so that a low Km may not necessarily imply a high affinity transport process. Gabapentin was negative for OCT like transport and LAT2 activity in the hCMEC/D3 and OCT1 transfected cells. Our data shows that gabapentin is a substrate for the influx transporter LAT1 at therapeutic concentrations.