title: Mutation of tyrosine 470 of human dopamine transporter is critical for HIV-1 Tat-induced inhibition of dopamine transport and transporter conformational transitions.
abstract: HIV-1 Tat protein plays a crucial role in perturbations of the dopamine (DA) system. Our previous studies have demonstrated that Tat decreases DA uptake, and allosterically modulates DA transporter (DAT) function. In the present study, we have found that Tat interacts directly with DAT, leading to inhibition of DAT function. Through computational modeling and simulations, a potential recognition binding site of human DAT (hDAT) for Tat was predicted. Mutation of tyrosine470 (Y470H) attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of this residue for Tat binding to hDAT. Y470H reduced the maximal velocity of [³H]DA uptake without changes in the K(m) and IC₅₀ values for DA inhibition of DA uptake but increased DA uptake potency for cocaine and GBR12909, suggesting that this residue does not overlap with the binding sites in hDAT for substrate but is critical for these inhibitors. Furthermore, Y470H also led to transporter conformational transitions by affecting zinc modulation of DA uptake and WIN35,428 binding as well as enhancing basal DA efflux. Collectively, these findings demonstrate Tyr470 as a functional recognition residue in hDAT for Tat-induced inhibition of DA transport and transporter conformational transitions. The consequence of mutation at this residue is to block the functional binding of Tat to hDAT without affecting physiological DA transport.
authors: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA.
authors: Narasimha M
authors: NM
authors: Midde
authors: Xiaoqin
authors: X
authors: Huang
authors: Adrian M
authors: AM
authors: Gomez
authors: Rosemarie M
authors: RM
authors: Booze
authors: Chang-Guo
authors: CG
authors: Zhan
authors: Jun
authors: J
authors: Zhu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D014443:Tyrosine / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C470431:tat peptide (1-72), Human immunodeficiency virus 1; D014443:Tyrosine; D004298:Dopamine
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