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 [3H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [3H]DA uptake and [3H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [3H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [3H]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.

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

“…Consistent with our previous reports3537, the current results show that mutations of His547 completely eliminate the inhibitory effect of Tat on DA transport, further suggesting that the Tat molecule is associated with DAT through intermolecular electrostatic attractions and complementary hydrophobic interactions. Our computational study predicts that the side chain of H547 forms a hydrogen bond with residue R49 of HIV-Tat and that the alanine mutation of H547 would be expected to change the local backbone conformation of hDAT-H547 and eliminate the hydrogen bond between hDAT-H547A/Tat-R49 (Fig.…”

“…Disrupting this motif will result in perturbation to the transport function, as evidenced by the decrease in V max in the mutants Y548H, Y470H, and Y551H. According to our previous work353637, the center residue of YYY motif, i.e., residue Y470, is critical for Tat-induced inhibition of DA uptake, which indicates that stability of YYY motif is also involved in hDAT/Tat binding. Thus, mutating tyrosine551 to histidine may not only change the transporter’s capacity for uptake but also inflict a structural change of hDAT/Tat binding.…”

“…1C) could stabilize the first part of transmembrane helix 10 (TM10a) by tying down residue Y470 with extracellular loop 6. Instability of TM10a could promote the formation of a salt bridge between D476 (in TM10a) and R85 (in TM1b); however, the D476-R85 salt bridge would close the “entrance gate” of DA353644454647. As a result, instability of TM10a is expected to block the DA entrance in the outward-open state, which may decrease the DA uptake efficiency of hDAT.…”

“…Interestingly, mutant H547P displays a dramatic decrease in DA uptake, which is accompanied by decreased total DAT expression (data not shown). Considering that site-directed mutagenesis studies on DAT typically result in a decrease in DA uptake35375758, the enhancement of DA transporter as evidenced by the H547A mutant suggests that targeting this residue may provide an exciting knowledge basis for the development of novel concepts for the therapeutic treatment of the HAND.…”

“…Through integrated computational modeling prediction and experimental validation, we have identified key residues in hDAT with which Tat interacts, which are critical for Tat-induced inhibition of DAT and transporter conformational transitions353637. Our studies provide mechanistic insights into identifying residues on DAT for Tat binding, which allows the exploration of the molecular targets on DAT for therapeutic interventions, improving neurocognitive function of HAND.…”