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

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⌽ trafficking motif (X is any residue and ⌽ 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 (Vmax) 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.

Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization

“…A modification in the conformation equilibrium could then lead to a concomitant change in the K m and V max values. A similar phenomenon has been observed in site-directed mutagenesis experiments on the serotonin transporter (hSERT) and the dopamine transporter (Loland et al 2002). Kristensen et al (2004) reported that two single mutations in the hSERT sequence increased the K m values that, in turn, led to an increase in the corresponding V max values.…”

The Second Intracellular Loop of the Glycine Transporter 2 Contains Crucial Residues for Glycine Transport and Phorbol Ester-induced Regulation

“…'' One possible explanation of the K422E phenotype, according to its kinetic behavior and its higher voltage dependence, would be the locking of the transporter in a conformational state that displays the cosubstrate-binding sites to the inside (''inward conformation'') (28). During the course of this work, Loland et al (31) proposed that the equivalent residue to Lys-422 in DAT (Lys-264) might form part of a conformationally active intracellular gating domain that might control access of the substrate to the intracellular milieu (31). If this hypothesis were correct also for GLYT2, residues that show alternating access and are extracellularly accessible in the outward-facing conformation would become occluded in the K422E background.…”

“…Although little is known about the three-dimensional structure of these transporters (16), site-directed mutagenesis approaches have permitted the identification of critical residues for the transport function in TMI, -III, and -IV (17)(18)(19)(20)(21)(22), which are candidates to contribute to the binding of substrates. In addition, conformationally active residues have been identified in several hydrophilic loops (23)(24)(25)(26)(27)(28)(29)(30), and it has been suggested that IL2-IL3-IL4 might be part of an intracellular "gating" domain, because a mutation of three residues in those loops of DAT seems to disrupt intramolecular interactions stabilizing the conformation able to bind extracellular substrate (31)(32)(33).…”

LeuT: A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure

“…133 Critical residues residing within the intracellular-facing half of the protein include an arginine and tryptophan in the N-terminal domain/TM1 of GAT1, 134 an asparate in TM8 of DAT, 135 and a tyrosine in the intracellular loop between TMs 6 and 7 of DAT. 136 The location and interactions of all of these residues in the LeuT structure reaffirm their postulated roles in the conformational changes that accompany substrate/ion binding and release. Near the extracellular "gate", at the bottom of a vestibule, is a water-mediated salt bridge between Asp404 (TM10) and Arg30 (TM1), the latter of which is structurally coupled to the substrate-binding site via a hydrogen bond network (Fig.…”