Cloning and gene silencing of LAT2, the L‐3,4‐dihydroxyphenylalanine (l‐DOPA) transporter, in pig renal LLC‐PK epithelial cells

ABSTRACT Organ‐specific overexpression of type 2 l‐amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SKR), accompanied by an enhanced ability to take up l‐DOPA, may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine‐mediated natriuresis. To understand the physiological role of LAT2‐mediated l‐DOPA handling, we used 21‐nucleotide small interfering RNA duplexes (siRNA) to specifically suppress LAT2 expression in LLC‐PK1 cells, a cell line that retains several properties of proximal tubular epithelial cells and takes up l‐DOPA largely through Na+‐independent transporters. After cloning the LLC‐PK1 LAT2 gene, one target region of LAT2 mRNA (nt 97‐117) was selected by scanning the length of the LAT2 gene for AA‐dinucleotide sequences and downstream 19 nucleotides. Levels of LAT2 cDNA, determined by real‐time quantitative RT‐PCR, were markedly (P