Mutational effects of the asparagine198 and glutamate223 residues on the human norepinephrine transporter on basal and HIV-1 Tat protein-induced inhibition of dopamine transport

Abstract

HIV-1 transactivator of transcription (Tat) protein induces dopaminergic dysregulation, which plays a central role in HIV-1-associated neurocognitive disorders. Computational modeling predicts that asparagine 198 and glutamate 223 of the human norepinephrine transporter (hNET) are key residues involved in Tat binding. This study investigated the effects of N198A and E223A mutations on basal and Tat-induced inhibition of dopamine (DA) uptake via hNET in CHO cells expressing WT hNET or its mutants. Compared to WT hNET, E223A mutation increased the affinity for nisoxetine and cocaine in inhibiting [³H]DA uptake. However, N198A and E223A decreased the affinity for cocaine inhibiting [³H]WIN35,428 binding, without altering the [³H]WIN35,428 binding under control. Kinetic analysis of [³H]DA uptake revealed that N198A and E223A did not alter the affinity for DA uptake but reduced the maximal velocity compared to WT hNET. An optimization study using recombinant Tat_1-86 at 0.25–140 nM revealed a K_i of 3.4 nM for inhibiting hNET-mediated DA uptake, with inhibition plateauing at above 8.75 nM. Treatment with 140 nM recombinant Tat_1-86 resulted in a 34 % reduction in [³H]DA uptake in WT hNET, which was attenuated in the N198A mutant but remained unchanged in E223A. However, the inhibition of [³H]DA uptake by 8.75 nM rTat_1-86 in WT hNET was attenuated in N198A and E223A. Moreover, N198A and E223A altered transporter conformational dynamics, as evidenced by changing the efflux of [³H]DA and [³H]MPP+. Collectively, these findings support the role of asparagine198 and glutamate223 as essential recognition residues in Tat-induced inhibition of DA uptake through hNET.