title: Functional analysis of pharmacogenetic variants of human organic cation/carnitine transporter 2 (hOCTN2) identified in Singaporean populations.
abstract: 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. Primary carnitine deficiency has been associated with a wide range of hOCTN2 gene mutations. Six novel nonsynonymous single nucleotide polymorphisms in the hOCTN2 gene were identified recently in Chinese and Indian populations of Singapore. The present study evaluated the impact of these polymorphisms on hOCTN2 function and expression in HEK-293 cells. Transport function was markedly impaired in variants that encoded amino acid substitutions D122Y (<20% of wild-type control) and K302E (∼45% of wild-type) in the large extracellular loop and large intracellular loop of hOCTN2, respectively. The function of the other four variants was unimpaired (E109K, V175M, K191N and A214V). From biotinylation and immunofluorescence experiments, 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 total cellular expression was unchanged. Transporter kinetic studies indicated a decrease in the V(max) for l-carnitine influx by K302E-hOCTN2 to 49% of wild-type control, while K(m) remained unchanged; kinetic evaluation of D122Y-hOCTN2 was not possible due to its low transport function. The K302E-hOCTN2 variant was also more susceptible than the wild-type transporter to inhibition by the drugs cimetidine, pyrilamine and verapamil. These findings indicate that impaired plasma membrane targeting of the D122Y and K302E-hOCTN2 variants that occur in Singaporean populations contributes to decreased carnitine influx.
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW 2006, Australia.
authors.forename: Dorothy Su Lin
authors.initials: DS
authors.lastname: Toh
authors.forename: Michael
authors.initials: M
authors.lastname: Murray
authors.forename: Kuan
authors.initials: K
authors.lastname: Pern Tan
authors.forename: Vishwaroop
authors.initials: V
authors.lastname: Mulay
authors.forename: Thomas
authors.initials: T
authors.lastname: Grewal
authors.forename: Edmund Jon Deoon
authors.initials: EJ
authors.lastname: Lee
authors.forename: Fanfan
authors.initials: F
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D044466:Asian People; D001692:Biological Transport; D019921:Biotinylation; D002331:Carnitine / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D027701:Organic Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D012846:Singapore; D000074058:Solute Carrier Family 22 Member 5; D044465:White People
chemical_list: D027701:Organic Cation Transport Proteins; C492020:SLC22A5 protein, human; D000074058:Solute Carrier Family 22 Member 5; D002331:Carnitine