Aspartate 345 of the Dopamine Transporter Is Critical for Conformational Changes in Substrate Translocation and Cocaine Binding

Abstract

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na ؉ /Cl ؊ -dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low V max and K m values for DA uptake, converted the inhibitory effect of Zn 2؉ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn 2؉ but not by Na ؉ . These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K ؉ substitution for Na ؉ . Thus, cocaine binding to D345N likely induces a different and Na ؉ -dependent conformational change, which may contribute to its Na ؉ -dependent uptake inhibitory activity.

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

“…In addition, mutant K422E displays functional features similar to the substitutions of residues Lys-264 (equivalent to Lys-422), Asp-345 in IL3, and Asp-436 in IL4 of DAT, proposed as a part of a network of molecular interactions needed for conformational changes during the transport cycle (32,33). Three lines of evidence allow us to conclude that K422E transporter molecules are more prone to acquire the inward-facing conformation.…”

“…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).…”

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

“…The ␤-subunit is an intracellular protein that contains two main domains, the Src homology 3 (SH3) and guanylate kinase (GK) domains. These domains, characterized by membrane-associated guanylate kinase, are known to allow modulation of LTCC activity and ␣ 1 -subunit trafficking (27,(57)(58)(59)(60)(61). The guanylate kinase domain contains the ␤-interaction domain, which binds directly to the ␣-interaction domain (AID) in the ␣ 1 -subunit (62,63).…”

FRET-based characterization of K264A, D345A, and Y335Amutants in the human dopamine transporter

“…Although uptake inhibition by inhibitors did not seem to be seriously affected by D345 mutants, all D345 mutants had extremely low V max and K m values for DA uptake (Chen et al, 2004). D345 possibly has a role in inhibitor binding and mutation of that residue converts DAT to a partially active state, which alters the interaction of cocaine analogs with the transporter (Chen et al, 2004). Although available data do not support direct contact between D345 and Y335, both residues have been suggested to be the active participants of a network of intermolecular connections taking place during substrate translocation.…”

“…D345, on the other hand, was characterized as an important element in both forward and reverse transport of DA. Although uptake inhibition by inhibitors did not seem to be seriously affected by D345 mutants, all D345 mutants had extremely low V max and K m values for DA uptake (Chen et al, 2004). D345 possibly has a role in inhibitor binding and mutation of that residue converts DAT to a partially active state, which alters the interaction of cocaine analogs with the transporter (Chen et al, 2004).…”