title: Mutations at tyrosine 88, lysine 92 and tyrosine 470 of human dopamine transporter result in an attenuation of HIV-1 Tat-induced inhibition of dopamine transport.
abstract: HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [(3)H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [(3)H]DA uptake and [(3)H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [(3)H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [(3)H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding.
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, SC, 29208, USA.
authors.forename: Narasimha M
authors.initials: NM
authors.lastname: Midde
authors.forename: Yaxia
authors.initials: Y
authors.lastname: Yuan
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Quizon
authors.forename: Wei-Lun
authors.initials: WL
authors.lastname: Sun
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Huang
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D014443:Tyrosine / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D010879:Piperazines; D054322:tat Gene Products, Human Immunodeficiency Virus; D014443:Tyrosine; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; C043425:vanoxerine; D003042:Cocaine; D008239:Lysine; D004298:Dopamine