Purpose: MCT10 activity has been shown to correlate to liver diseases, including nonalcoholic steatohepatitis (NASH)1, and has been shown to be essential for aromatic amino acid homeostasis control2. The purpose of this research was to develop and validate an assay for MCT10 in MDCK-II cells to allow detailed in vitro characterization of this transporter. Methods: MDCK-II cells were seeded on Millipore Millicell 96-well insert plates (PCF – 0.4 µm), then transfected using OPTI-EXPRESSIONTM technology with plasmids either encoding MCT10 or a control vector. In addition, various accessory proteins were co-transfected. Assays were conducted 48 hours after transfection to allow the appropriate localization of the transporter in polarized MDCK-II cells. A variety of amino acid substrates such as tyrosine, phenylalanine, levodopa, and gabapentin were tested for potential apical uptake, basolateral uptake, and efflux. Substrates were also applied in one chamber and transcellular flux was measured. Results: Initial evaluations revealed lower intracellular accumulation in MCT10-transfected cells than in control cells, no matter whether amino acid substrates were applied to the apical surface or the basal surface of the MDCK-II cells, suggesting that MCT10 may function as an efflux transporter rather than an uptake transporter. By applying the substrates to the basal surface and measuring transcellular flux into the apical chamber, it was possible to establish a robust assay for measuring MCT10 activity. Tyrosine, phenylalanine, and levodopa were all shown to be strong substrates of MCT10-dependent transcellular flux, with ~5-fold increases in P<sub>app(B- >A) in cells expressing MCT10 compared to control cells. Gabapentin also appears to be transported by MCT10. Transcellular transport appears to be linear for at least 90 minutes and aromatic amino acids appear to be relatively low affinity substrates. Conclusion: This novel transcellular transport assay provides an improved method for in vitro characterization of the MCT10 transporter. Since MCT10 appears to be important for maintaining circulating levels of amino acids as well as thyroid hormones3, and has been linked to liver diseases, it may be a potential drug target in disease states. References: 1. Lake et al., 2015. Amino Acids 47:603-615. 2. Mariotta et al., 2012, J. Physiol. 590:6413-6424. 3. Felmlee et al., 2020, Pharmacol. Rev. 72:466-485.
