Abstract
Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT.
Highlights
Despite the widespread use of efficacious antiretroviral therapies to control peripheral human immunodeficiency virus (HIV) infection and improve the life of HIV patients, HIV-associated neurocognitive disorders (HAND) remain highly prevalent and represent a significant health problem[1]
Post hoc analysis indicated that cocaine IC50 values were significantly decreased in Y470H-human DAT (hDAT) (196 ± 23 nM, least significant difference (LSD): p < 0.001) and Y88F-hDAT (157 ± 10 nM, p < 0.001) but not in Y470F-hDAT (281 ± 16 nM), compared to the wild type (WT)-hDAT (324 ± 19 nM)
The current study reports the allosteric modulatory effects of SRI-compounds on cocaine and Tat binding to hDAT
Summary
Mutations of Y470 and Y88 attenuate effects of SRI-20041 on cocaine-induced inhibition of DAT activity. Y470H-hDAT displayed a decrease in the dissociation rate (K−1 = 0.068 ± 0.015 min−1, p < 0.001) compared to WT, indicating that mutation of Y470 alters cocaine-induced dissociation of [3H]WIN35,428 binding. The effect of SRI-30827 on cocaine-induced dissociation of [3H]WIN35,428 binding was attenuated in Y470H-hDAT (K−1 = 0.061 ± 0.027 min−1, t(7) = 0.23, p = 0.82) and Y88F-hDAT (K−1 = 0.095 ± 0.014 min−1, t(8) = 1.69, p = 0.13), respectively, relative to their respective controls (cocaine alone). To determine whether the Tat-induced inhibition of DAT binding sites is mediated through an allosteric modulation manner, the ability of SRI-30827 to attenuate the inhibitory effect of Tat was determined (Fig. 4). SRI-30827 itself did not alter the specific [3H]WIN35,428 binding (p > 0.05); SRI-30827/Tat showed no inhibitory effect on the specific [3H]WIN35,428 binding compared to SRI30827 alone, indicating that SRI-30827 attenuates Tat-induced decrease in DAT binding sites
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