FIGURE 4.

Exchange of glycine 478 by cysteine in rOct1 decreases the turnover for MPP⁺. FLAG-tagged rOct1 wild type (rOct1-FLAG) or mutant (rOct1(G478C)-FLAG) were expressed in oocytes. A, plasma membranes of the oocytes were isolated, and the proteins were separated by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue or analyzed in a Western blot using an anti-FLAG antibody. The densities of the antibody-stained bands were quantified by densitometry. Mean values ± S.E. of three experiments are indicated. B, comparison of V_max values of MPP⁺ uptake mediated by rOct1-FLAG and rOct1(G478C)-FLAG. Uptake of 0.1 μm [³H]MPP⁺ in oocytes expressing rOct1-FLAG or rOct1(G478C)-FLAG or in noninjected oocytes were measured, and the transporter expressed uptake rates were calculated. The V_max values were calculated according to the Michaelis-Menten equation using the K_m values that were determined in separate experiments (rOct1-FLAG 7.75 ± 1.17 μm and rOct1(G478C)-FLAG 5.23 ± 0.61 μm (n = 3 each)). Mean values ± S.E. are indicated. The number of analyzed oocytes is indicated in parentheses. Three independent experiments using different oocytes batches were performed. The uptake rates were normalized to the mean values of MPP⁺ uptake in rOct1-FLAG-expressing oocytes of the respective experiment. ***, p < 0.001. n.s., not significant.