GFP-human high-affinity carnitine transporter OCTN2 protein: Subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect

Individuals with the plasmalemmal high-affinity carnitine transporter defec t present with progressive infantile-onset carnitine-responsive cardiomyopa thy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopa thy, and failure to thrive. The carnitine uptake defect (CUD) has been docu mented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts, The cDNA encoding the high-affinity sodium-dependent human carnitine transp orter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expressio n studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lin es with the CUD, Transfection of cell lines from 12 unrelated patients (nin e fibroblast and three lymphoblastoid) with a GFP construct harboring the w ildtype full-length OCTN2 cDNA was done using Lipo-TAXI. Transient and stab le expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable tran sfection of the lymphoblastoid cell, lines were achieved. There was functio nal restoration of carnitine uptake in the transfected mutant cell lines, t hereby confirming the identity of the transfected cDNA. In addition, we rep ort the first demonstration of the subcellular localization of an in-frame fusion GFP-human high-affinity carnitine transporter OCTN2 protein in the p lasma membrane by confocal laser-scanning fluorescence microscopy. (C) 1999 Academic Press.