[Establishment and application of cell models with stable expression of hOCTN1/2]

Human carnitine/organic cation transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1 (+) plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K(m) and V(max) of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 μmol·L-1 and 1 427 ± 49 pmol·mg(-1)(protein)·min(-1); while K(m) and V(max) of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 μmol·L(-1) and 2 454 ± 64 pmol·mg(-1)(protein)·min(-1). Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.