Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

Pamela M Quizon,Jun Zhu,Narasimha M Midde,Chang-Guo Zhan,Yaxia Yuan,Wei-Lun Sun

doi:10.1038/srep39048

Pamela M Quizon, Jun Zhu + Show 4 more

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https://doi.org/10.1038/srep39048

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Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

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An estimated thirty-four million people worldwide are living with HIV
The hydrogen bond with the H547 side chain is expected to be broken with the His547 [Histidine to Alanine (H547A) mutation, which is consistent with decreased inhibitory activity of Tat on Human DAT (hDAT)-H547A
According to the molecular dynamics (MD) simulation and the potential of main force (PMF) energy calculation performed in our previous work[36], we found that the H547A mutation could enhance the strength of the YYY motif

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Introduction

An estimated thirty-four million people worldwide are living with HIV. More than 50% of HIV-1 positive individuals suffer from neurological complications collectively referred to as HIV-1-associated neurocognitive disorders (HAND)[1]. Considering the progressive and neurodegenerative nature of HAND, establishing an early intervention strategy would be beneficial to the preservation of neurocognitive function in HIV-infected individuals. HIV-induced elevated levels of extracellular DA in the CNS can stimulate viral replication in human macrophages within DA-rich brain regions[15,16,17], resulting in viral protein release. Human DAT (hDAT) activity is strikingly reduced in HIV-1-infected cocaine-using patients, correlating with the severity of HIV-1 associated cognitive deficits[5,6]. By producing oxidative stress-induced damage to dopaminergic neurons, prolonged exposure to Tat protein eventually causes DAT-mediated dysregulation of DA to accelerate the progression of HAND11. Our recent computational modeling study demonstrated that mutation of histidine to alanine at hDAT 547 (H547A) increases DA transport and attenuates Tat inhibitory effect on DAT function[38]. We evaluated the functional influence of other substitutions of Histidine[547] (His547) and its associated residues, tyrosine[548] and tyrosine[551], in DA transport as well as Tat-induced inhibition of DA uptake

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