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. 2019 Mar 7;9:3843. doi: 10.1038/s41598-019-39872-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of Y88F/H547A and Y88F/K92M/H547A on [³H]DA uptake and [³H]WIN35,428 binding. (A) Kinetic parameters (V_max and K_m) of [³H]DA uptake in WT hDAT and mutants. [³H]DA uptake assay was conducted in PC12 cells transfected with WT hDAT and mutants by incubation with one of 6 mixed concentrations of the [³H]DA as the total rate of DA uptake. Nonspecific uptake of [³H]DA was determined by a parallel incubation of each mixed DA concentrations with nomifensine (10 µM, final concentration). Specific [³H]DA uptake (V_max) was calculated by subtraction of nonspecific DA uptake from total DA uptake. The parameter values were presented as means ± S.E.M. *p < 0.05 compared to WT hDAT value (unpaired Student’s t test) (n = 4–5/group). (B) Saturation analysis of [³H]WIN35,428 binding in WT hDAT and mutants. B_max and K_d values were estimated by fitting one-site binding and presented as means ± S.E.M. *p < 0.05 compared to WT hDAT value (unpaired Student’s t test) (n = 4–12/group).