Functional analysis of novel variants in the organic cation/ergothioneine transporter 1 identified in Singapore populations.

The human organic cation/ergothioneine transporter 1 (hOCTN1, gene symbol SLC22A4) is responsible for the cellular uptake of substances, such as L-ergothioneine, which is an important antioxidant in mammalian cells. The common-function-altered variant L503F-hOCTN1 has been associated with susceptibility to Crohn's disease in certain populations. Previously, we identified eight novel nonsynonymous single-nucleotide polymorphisms (SNPs) in the SLC22A4 gene in the Chinese and Indian populations of Singapore. The present study evaluated the impact of these novel SNPs on hOCTN1 transport function in HEK-293 cells. Transport uptake assays with L-ergothioneine were used to assess the function of the variant transporters. Cell surface biotinylation and Western blot analysis were used to characterize cellular transporter expression. Comparative modeling was used to locate amino acid substitutions in the topology of hOCTN1 in order to account for altered transport function. Transporter activity was markedly impaired in four of the naturally occurring hOCTN1 variants (R63H, R83P, G482D, and I500N). Multiple glycosylated isoforms of hOCTN1 proteins were identified in the plasma membrane and in the whole cell. Either the total cellular or membrane expression of the functionally deficient transporter variants was lower than that of the wild-type hOCTN1. The underlying mechanism involves both impaired transporter-substrate binding affinity and turnover rate. Considered together, several naturally occurring SNPs in the SLC22A4 gene encode variant hOCTN1 transporters that may impact the cellular uptake of L-ergothioneine and other substrates, with the potential to influence the antioxidant capacity of human cells.

Functional analysis of pharmacogenetic variants of human organic cation/carnitine transporter 2 (hOCTN2) identified in Singaporean populations.

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.