Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Abstract

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT Aa ) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln 332 in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln 332 to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters. The serotonin (5-hydroxytryptamine (5-HT)2 ) transporter (SERT) is a member of the neurotransmitter sodium symporters (NSSs) (also referred as Na ϩ /Cl Ϫ -dependent transporters), whose function is to return 5-HT into presynaptic neurons after release into the synapse (1-3). Other family members include the transporters for dopamine (DAT), norepinephrine, ␥-aminobutyric acid, and glycine (1-4). These transporters are of particular interest as targets for many drugs, including antidepressants, anticonvulsants, and abused psychostimulants, such as cocaine and amphetamines (3). Although these transporters may share a common topology of 12 transmembrane ␣-helices (TMHs), intracellular N and C termini, and a large extracellular loop between TMHs III and IV with putative glycosylation sites, actual structural information for these proteins is limited.A high resolution crystal structure for members of the human neurotransmitter transporter family has yet to be solved, impeding the study of these important drug targets. A breakthrough came when the crystal structure of a bacterial homologue of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters was determined (5). The leucine transporter (LeuT Aa ) from the bacteria Aquifex aeolicus was crystallized with the substrate and two Na ϩ ions occluded in the structure (5). It shares 20 -25% of overall sequence identity with the eukaryotic neurotransmitter transporters, including clusters of high sequence conservation (5). Our group and others have supported the use of LeuT Aa as an appropriate template for ho...

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

“…Cocaine has been shown to stabilize an outward-facing conformation of SERT, and thus increases accessibility of residues in the extracellular permeation pathway. Accordingly, and consistent with previous findings (44), cocaine increased the accessibility of Q332C (Fig. 9).…”

“…Cocaine and ibogaine have been shown to stabilize outwardand inward-facing SERT conformations, respectively, as demonstrated by their ability to increase or decrease accessibility of positions in the extracellular or cytoplasmic permeation pathways (12,40,(42)(43)(44). However, much less is known about the conformational state of SERT that is stabilized by most other SERT inhibitors.…”

“…However, escitalopram, talopram, and atomoxetine have been shown to be competitive inhibitors of SERT (8,23,46), indicating that their binding site overlaps the central substrate binding site. Thus, rather than blocking access to Q332C, which is located outside the central S1 site (44), it seems more likely that the inhibitors decrease the reactivity of this residue by stabilizing an occluded-or inward-facing conformation.…”

“…We therefore selected four inhibitors, atomoxetine, escitalopram, nisoxetine, and talopram, that were highly sensitive to the L406E mutation (Ͼ18-fold gain-of-potency) in addition to ibogaine and cocaine, which were less sensitive to the mutation (Ͻ6-fold gain-of-potency), and determined the ability of the inhibitors to modify the reactivity of Q332C in the extracellular permeation pathway. The Q332C mutant (generated in the C109A background that is insensitive to externally applied MTS reagents (45)) has previously been shown to be accessible to MTSET only when the transporter resides in an outward-facing conformation (44). COS7 cells expressing the C109A/Q332C mutant were preincubated with MTSET in the absence or presence of increasing inhibitor concentrations.…”

Dopamine Transport by the Serotonin Transporter: A Mechanistically Distinct Mode of Substrate Translocation

“…Recently, Torres-Altoro et al described an hSERT mutant, Q332C, the transport activity of which was susceptible to MTS reagents. Moreover, coincubation with 5HT hindered MTS inactivation of transport by Q332C whereas cocaine coincubation facilitated it, suggesting that these ligands differentially alter the MTS accessibility of the residue and conformation of the protein (Torres-Altoro et al, 2010). Thus, we were interested to test whether DA changes SERT conformation in a manner different from 5HT.…”