Impact of disruption of secondary binding site S2 COMMUNICATION on dopamine transporter function

The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S-2) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S-1). The present experiments are aimed at disrupting S-2 by mutating Asp476 and Ile 159 to Ala. Both mutants displayed a profound decrease in [H-3]DA uptake compared with wild-type associated with a reduced turnover rate k(cat). This was not caused by a conformational bias as the mutants responded to Zn2+ (10 mu M) similarly as WT. The dopamine transporters with either the D476A or 1159A mutation both displayed a higher K-i for dopamine for the inhibition of [H-3](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S-1 and S-2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters.