P328 Functional Analysis of Pharmacogenetic Variants of Human Organic Cation/Carnitine Transporter 2 (hOCTN2) Identified in Singaporean Populations

Dorothy Su Lin Toh , Pharmacy, University of Sydney, Sydney, Australia
Michael Murray , Pharmacy, University of Sydney, Sydney, Australia
Tan Kuan Pern , Bioinformatics Institute, Agency for Science, Technology and Research, Singapore
Edmund Lee , Department of Pharmacology, National University of Singapore, Singapore, Singapore
Fanfan Zhou , Pharmacy, University of Sydney, Sydney, Australia
The human organic cation/carnitine transporter-2 (hOCTN2; SLC22A5) mediates the cellular influx of organic cations such as carnitine, which is essential for fatty acid oxidation, and a number of cationic drugs. Primary carnitine deficiency has been associated with a wide range of hOCTN2 gene mutations that may encode functionally defective hOCTN2 transporter variants. Previously we identified six novel nonsynonymous single nucleotide polymorphisms in the SLC22A5 gene in individuals from Chinese and Indian populations of Singapore1. The present study evaluated the impact of these polymorphisms on hOCTN2 function and expression in transfected HEK-293 cells. Transport function with the substrates L-carnitine and tetraethylammonium was markedly impaired in variants that encoded amino acid substitutions D122Y (<20% of wild-type control) and K302E (~45% of wild-type); these amino acid substitutions are located respectively in the large extracellular and large intracellular loops of hOCTN2. In contrast, the function of the other four variants was unimpaired (E109K, V175M, K191N and A214V). Subsequent biotinylation and immunofluorescence experiments indicated that the expression of the D122Y and K302E-hOCTN2 variants at the plasma membrane of HEK-293 cells was decreased relative to the wild-type hOCTN2, but that total cellular expression was unchanged. This is also consistent with findings from transporter kinetic studies that the Vmax for L-carnitine uptake was decreased in K302-hOCTN2 to ~50% of wild-type control, while Km remained unchanged; kinetic evaluation of D122Y-hOCTN2 was not possible due to variable rates of transport function at low L-carnitine concentrations. The K302E-hOCTN2 variant was also more susceptible than the wild-type transporter to inhibition by the drugs cimetidine, pyrilamine and verapamil. Considered together these findings indicate that impaired plasma membrane maturation of the previously unidentified D122Y and K302E-hOCTN2 variants that occur in Singaporean populations contributes to decreased carnitine influx.

1D.S.L. Toh, J.Y. Yee, S.H. Koo, M. Murray and E.J.D. Lee, Genetic Variations of the SLC22A5 Gene in the Chinese and Indian Populations of Singapore. Drug Metab Pharmacokinet (2010) 25: 112-119