TY - JOUR
T1 - Mutational effects of human dopamine transporter at tyrosine88, lysine92, and histidine547 on basal and HIV-1 Tat-inhibited dopamine transport
AU - Sun, Wei Lun
AU - Quizon, Pamela M.
AU - Yuan, Yaxia
AU - Strauss, Matthew J.
AU - McCain, Richard
AU - Zhan, Chang Guo
AU - Zhu, Jun
N1 - Funding Information:
Research reported in this publication was supported by National Institutes of Health grants to JZ (R01DA035714 and R21DA041932).
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the V max values of [ 3 H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [ 3 H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [ 3 H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.
AB - Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the V max values of [ 3 H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [ 3 H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [ 3 H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.
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U2 - 10.1038/s41598-019-39872-1
DO - 10.1038/s41598-019-39872-1
M3 - Article
C2 - 30846720
AN - SCOPUS:85062586463
VL - 9
IS - 1
M1 - 3843
ER -
