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Impact of disruption of secondary binding site S 2 on dopamine transporter function
Author(s) -
Zhen Juan,
Reith Maarten E. A.
Publication year - 2016
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13704
Subject(s) - dopamine transporter , transporter , dopamine , dopamine plasma membrane transport proteins , allosteric regulation , binding site , chemistry , neurotransmitter transporter , tropane , biochemistry , stereochemistry , biology , enzyme , neuroscience , gene
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 Ile159 to Ala. Both mutants displayed a profound decrease in [ 3 H] 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 Zn 2+ (10 μM) similarly as WT . The dopamine transporters with either the D476A or I159A mutation both displayed a higher K i for dopamine for the inhibition of [3H](−)‐2‐β‐carbomethoxy‐3‐β‐(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.X‐ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S 2 in the extracellular vestibule proximal to the primary binding site S 1 which is closely linked to one of the Na + binding sites. This work examines the relationship between S 2 and S 1 sites. We found that S 2 site impairment severely reduced DA transport and allosterically reduced S 1 site affinity for the cocaine analog [ 3 H]CFT. Our results are the first to lend direct support for the application of the two‐site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA 1 ) to the primary S 1 site (along with Na + ), binding of a second DA (DA 2 ) to the S 2 site triggers, through an allosteric interaction, the release of DA 1 and Na + into the cytoplasm.