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. 2013 Sep 23;288(45):32160–32171. doi: 10.1074/jbc.M113.502971

FIGURE 1.

FIGURE 1.

Gly308 functions as a pivotal molecular hinge required for TBZ binding. A, replacement of Gly308 by Ala (G308A) confers resistance to TBZ in yeast cells, whereas a Pro replacement rescues TBZ-resistant phenotype. ADU1–7 cells transformed with pAES426 (empty vector) or pAES426 harboring either rVMAT2 or Gly308 mutants were grown in minimal medium and diluted to comparable densities. 5 μl of serial dilutions of the culture were spotted on rich solid medium (YPD) supplemented with 1.5 mm MPP+ (upper panel), or 40 μm acriflavine (lower panel). Where indicated, the plates contained 2 μm TBZ. Growth was analyzed after 48 h at 30 °C. The plates are representative of at least three independent experiments. B, time course of [3H]serotonin ([3H]5HT) transport by Gly308 mutants reconstituted in proteoliposomes. The uptake assay was performed as described under “Experimental Procedures.” Ammonium-loaded proteoliposomes (1 μl) were diluted into 200 μl of reaction buffer to generate the pH gradient that drives serotonin transport and 50 nm valinomycin was added to prevent the generation of a membrane potential by the electrogenic exchange of 2H+ with one serotonin molecule. All data are mean ± S.E. of 2–3 experiments. C, multiple sequence alignment of members of the SLC18 family in the region of residues 304–312 (TM7). Multiple alignment was performed using Clustal Omega (38). A conservation logo was created using WebLogo 3.3 (39, 40). Only a fraction of the alignment corresponding to residues 304–312 is shown. D, kinetic properties of rVMAT2 and Gly308 mutants. Proteoliposomes were prepared from HEK293 cells expressing either rVMAT2 or Gly308 mutants. Protein determination and serotonin uptake in proteoliposomes and [3H]TBZOH binding were performed as described under ”Experimental Procedures.“ The “specificity constant” Vmax/Km was obtained by simply dividing the corresponding values given in the table and the units are those shown. TBZ sensitivity was assessed by calculating the amount of ligand required to inhibit serotonin transport by 50% (IC50). The uptake assay was performed in the presence of increasing concentrations of TBZ (0–200 nm) for 20 min. Reserpine sensitivity was assessed by calculating the concentration required to inhibit serotonin transport by 50% (IC50) (0–100 nm).