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

Abstract

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.

Mutational effects of human dopamine transporter at tyrosine88, lysine92, and histidine547 on basal and HIV-1 Tat-inhibited dopamine transport

“…Based on the constructed hDAT-Tat binding model in our previous work 20 , hDAT residues Y88, K92, and H547 could form hydrogen bonds with residues K19, P18, and R49 of HIV-1 Tat, respectively. According to our previous computational and experimental results, single mutation of either hDAT Y88 (Y88F), K92 (K92M) or H547 (H547A) could significantly attenuate the binding between DAT and Tat 17,18 . As shown in Fig.…”

“…Using computational modeling and experimental approach, we have identified several key residues on human DAT (hDAT), which are crucial for Tat-hDAT interaction and dynamic transport process 9 . Furthermore, we have demonstrated that in vitro exposure to Tat reduced reuptake of DA via hDAT in cells 16–18 and rat striatal synaptosomes 19 . The inhibitory effect of Tat on DAT function results from Tat directly interacting with DAT 16,20,21 .…”

“…The inhibitory effect of Tat on DAT function results from Tat directly interacting with DAT 16,20,21 . Single point mutations of hDAT at tyrosine88 (to phenylalanine, Y88F), lysine 92 (to methionine, K92M), histidine547 (to alanine, H547A) differentially alter basal DA uptake but attenuate the Tat inhibitory effects on DA transport 17,18 . For example, DA uptake is decreased in K92M and increased in H547A, respectively; however Y88F mutant preserves basal DA uptake 17,18 .…”

“…Single point mutations of hDAT at tyrosine88 (to phenylalanine, Y88F), lysine 92 (to methionine, K92M), histidine547 (to alanine, H547A) differentially alter basal DA uptake but attenuate the Tat inhibitory effects on DA transport 17,18 . For example, DA uptake is decreased in K92M and increased in H547A, respectively; however Y88F mutant preserves basal DA uptake 17,18 . Notably, the mutational effects on normal DA uptake and Tat inhibitory effect on DAT function are associated with alterations of transporter conformational transitions 9 .…”

Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS

“…When examining the neurotoxic effects of Tat it is also important to consider the role of disruptions in healthy dopamine signaling. A potential alternative/complementary explanation for the effects that we have observed stems from Tat’s ability to bind and inhibit dopamine transporter activity (DAT) (Quizon et al, 2016) leading to increased dopamine levels which could contribute to neurotoxic conditions. Interestingly, human DAT activity in HIV-1 patients negatively correlates with HAND symptom severity (Wang et al, 2004; Chang et al, 2008) suggesting it plays an important role in the development of HAND pathology.…”