Abstract
Binding of Zn(2+) to the endogenous Zn(2+) binding site in the human dopamine transporter leads to potent inhibition of [(3)H]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn(2+) switch into an activating Zn(2+) switch, allowing Zn(2+)-dependent activation of the transporter. The tyrosine is part of a conserved YXX Phi trafficking motif (X is any residue and Phi is a residue with a bulky hydrophobic group), but Y335A did not show alterations in surface targeting or protein kinase C-mediated internalization. Despite wild-type levels of surface expression, Y335A displayed a dramatic decrease in [(3)H]dopamine uptake velocity (V(max)) to less than 1% of the wild type. In addition, Y335A showed up to 150-fold decreases in the apparent affinity for cocaine, mazindol, and related inhibitors whereas the apparent affinity for several substrates was increased. However, the presence of Zn(2+) in micromolar concentrations increased the V(max) up to 24-fold and partially restored the apparent affinities. The capability of Zn(2+) to restore transport is consistent with a reversible, constitutive shift in the distribution of conformational states in the transport cycle upon mutation of Tyr-335. We propose that this shift is caused by disruption of intramolecular interactions important for stabilizing the transporter in a conformation in which extracellular substrate can bind and initiate transport, and accordingly that Tyr-335 is critical for regulating isomerization between discrete states in the transport cycle.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Proceedings of the National Academy of Sciences of the United States of America |
| Vol/bind | 99 |
| Udgave nummer | 3 |
| Sider (fra-til) | 1683-8 |
| Antal sider | 6 |
| ISSN | 0027-8424 |
| DOI | |
| Status | Udgivet - 5 feb. 2002 |
Fingeraftryk
Dyk ned i forskningsemnerne om 'Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle'. Sammen danner de et unikt fingeraftryk.Citationsformater
Loland, C. J., Norregaard, L., Litman, T., & Gether, U. (2002). Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1683-8. https://doi.org/10.1073/pnas.032386299