CHARACTERIZATION OF THE HUMAN PLASMALEMMAL CARNITINE TRANSPORTER IN CULTURED SKIN FIBROBLASTS

Carnitine is an essential cofactor for long-chain fatty acid oxidation . We characterized the human carnitine transporter in vitro in a cultu red skin fibroblast model both at the previously established K-m conce ntration of carnitine uptake in fibroblasts (5 mu mol/liter) and at 0. 05% K-m (0.25 mu mol/liter). A rapid exponential dose-dependent decrea se in mean percentage of carnitine uptake was demonstrated with increa sing concentrations of nigericin, but no significant decrease was foun d with equimolar amounts of valinomycin. This would suggest that the N a+ gradient is integral to carnitine transport function. Interference of the Na+ (out-in) gradient by nigericin may be secondary to cytoplas mic acidification by this K+ proton ionophore. The rate of uptake was fully saturated at an extracellular Na+ concentration of 150 mmol/lite r. Replacement of 150 mmol/liter extracellular Na+ with Li+ resulted i n an 80 and a 50% reduction, and replacement with K+ and Rb+ ions resu lted in a 100 and am 85 to 90% reduction in carnitine uptake, respecti vely, at carnitine concentrations of 0.25 and 5 mu mol/liter, underlin ing the specific requirement for the Na+ ion. The effects of different site-specific respiratory chain toxins, namely, rotenone (complex I), antimycin A (complex III), and potassium cyanide (KCN) (complex IV) o n carnitine uptake was also examined. A rapid exponential dose-depende nt decrease in mean percentage of carnitine uptake with increasing con centrations of inhibitors was demonstrated. These data suggest either a metabolic energy requirement of the carnitine transporter or interfe rence of the Na+ (out-in) gradient by a proton gradient (in-out) secon dary to the accumulation of intracellular H+ ions, due to the action o f the respiratory chain toxins, further suggesting that the transporte r is sensitive to and inhibited by intracellular H+ ions. The effects of several sulfhydryl-binding agents, namely 2,4-dinitrofluorobenzene, N-ethylmaleimide, and mersalyl acid, were examined, and a significant inhibition of carnitine uptake was demonstrated, suggesting that free sulfhydryl groups are also integral to the import function of the hum an fibroblast transporter. (C) 1996 Academic Press, Inc.