Extended Data Fig. 1: Functional characterization and purification of hDAT.

a, Representative HPLC profiles of full-length hDAT (DAT^WT) and hDAT^EM in the LMNG detergent buffer. b, Kinetic DA uptake activities of DAT^EM as compared to DAT^WT. Data represented as mean ± S.E.M. (n = 3 biological independent experiments) and curve has been normalized to the surface expression level (c). The K_m and V_max value are 0.66 ± 0.16 μM, 2243 ± 108.8 CPM and 0.83 ± 0.43 μM, 1957 ± 214.9 CPM for DAT^WT and DAT^EM, respectively. There is no significant difference in the comparison of K_m (P = 0.7328) and V_max (P = 0.3003) using two-sided unpaired t-test. c, Cell surface biotinylation of DAT^WT and DAT^EM in HEK293F cells. The samples of whole cell lysate (lane 1) and surface protein (lane 2–4) are labeled with biotin and recovered using streptavidin beads. No protein was detected in unbiotinylated cell lysate sample (lane 5). The experiments were repeated three times, yielding similar results. d-f, Concentration-inhibition curves of [³H]DA uptake by MPH (d), GBR12909 (e) and BZT (f) for DAT^WT and DAT^EM. Data represent the mean ± S.E.M. (n = 3 biological independent experiments). The [³H]DA uptake is normalized to the value measured in the absence of investigated drugs. There is no significant difference between the IC₅₀ values of MPH, GBR12909 and BZT for DAT^WT and DAT^EM, supported by P-values of 0.4723, 0.6728 and 0.9161, respectively, calculated by two-sided unpaired t-test. g, The melting curve of DAT^EM in 0.1% LMNG in comparison to DAT^EM in 0.1% DDM. h, Size exclusion chromatography (SEC) profiles of purified DAT^EM in the LMNG buffer and in MSP1D1E3 nanodiscs. i, The Coomassie-stained SDS-PAGE gel of the DAT^EM–MSP1D1E3 cryo-EM samples. The experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.

Source Data