Table 1.

Kinetic parameters of carnitine counterflow of CaiT bearing substitutions of given amino acids.

CaiT variant	Km [µM]	Vmax [µmol min−1 mg−1]
wild-type	45.3 ± 6.9	2.13 ± 0.08
D288A	114.1 ± 17.9	1.86 ± 0.09
D288R	not detectable	<3% of wild-type

Carnitine counterflow was measured with proteoliposomes reconstituted with purified CaiT (lipid to protein ratio of 100:1 (w/w)) as described¹. The proteoliposomes were preloaded with 10 mM unlabeled L-carnitine at 4 °C overnight. Counterflow was initiated by 200 to 800fold dilution of preloaded proteoliposomes into buffer containing L-[methyl-¹⁴C]carnitine at final concentrations ranging from 0.014 to 1 mM at 25 °C. Proteoliposomes not preloaded with L-carnitine served as negative control, and resulting transport rates were used for correction. The data were fitted to the Michaelis-Menten equation and replotted according to Lineweaver-Burk and Eadie-Hofstee using the kinetic module of the SigmaPlot software or GraphPad Prism. Shown are mean values and standard deviations calculated from three independent experiments.