Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK1 epithelial cells

Organ-specific overexpression of type 2 L-amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SHR), 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