Abstract
The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs.
Highlights
Potentially be exploited in the development of treatments of stimulant abuse, and several DAT inhibitors have been pursued as pharmacotherapies for cocaine addiction[9]
We examined the role of non-conserved residues within the S1 site of human SERT and hNET for inhibitor selectivity by mutating all non-conserved residues to the corresponding residues in the other transporter, and thereby in principle transferred the S1 site from hNET into hSERT and vice versa
We took advantage of the high-resolution structural insight into human monoamine transporters (MATs) that has become available via the x-ray crystal structures of Drosophila melanogaster DAT (dDAT) to examine the role of non-conserved residues in the extracellular entrance pathway and within the central S1 binding site for inhibitor selectivity in hNET and hDAT
Summary
Potentially be exploited in the development of treatments of stimulant abuse, and several DAT inhibitors have been pursued as pharmacotherapies for cocaine addiction[9]. Current structural understanding of human MATs is based on x-ray crystal structures of bacterial and invertebrate homologs, which include the bacterial amino acid transporters LeuT and MhsT and the Drosophila melanogaster DAT (dDAT)[10,11,12,13] These structures have established that MATs share a conserved topology consisting of 12 transmembrane domains (TMs) arranged in a barrel-like bundle with the substrate binding site (denoted the S1 site) located in the core of the protein structure (Fig. 1). It was recently demonstrated that some inhibitors have different rates of binding (association) to DAT and SERT whereas their rates of unbinding (dissociation) were similar, indicating that diffusion of inhibitors through the entry pathway contribute to MAT inhibitor selectivity[44] Notable, this is in analogy to G protein-coupled receptors, where binding of certain ligands is influenced en route to the binding site by residues in distal permeation sites close to the receptor surface[45,46]. Our findings provide new insight into the molecular basis for inhibitor selectivity between hNET and hDAT, which may be used in the rational design and development of novel therapeutic agents targeting hNET and hDAT with tailor-made selectivity profiles
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