Helix XI contributes to the entrance of the serotonin transporter permeation pathway

Abstract

The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP + ). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP + transport. We examined the importance of TMH XI residues F551 and F556 for MPP + translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP + transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP + transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP + was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT Aa ) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.Keywords: cocaine; antidepressant; amphetamine; biogenic amine; neurotoxinThe neurotoxin 1-methyl-4-phenylpyridinium (MPP + ) is translocated by the neurotransmitter transporters for dopamine, norepinephrine, and serotonin (DAT, NET, and SERT, respectively) (Javitch et al. 1985;Paczkowski et al. 1999;Sitte et al. 2000Sitte et al. , 2001Martel and Keating 2003). These transporters belong to the Na + /Cl À -dependent family of transporters that contain 12 transmembrane helices (TMHs) with intracellular N and C termini. Previous studies of DAT identified residues important for MPP + translocation in TMHs I, VII, and XI (Kitayama et al. 1992(Kitayama et al. , 1993Mitsuhata et al. 1998). Simultaneous mutation of TMH XI residues S527, Y533, and S538 to alanine in rat DAT (rDAT) resulted in enhanced MPP + uptake compared to wild-type rDAT (Kitayama et al. 1993). These mutations exerted modest effects on dopamine uptake but had little impact on cocaine analog affinity (Kitayama et al. 1993). Although that study suggests determinants in TMH XI of rDAT important for MPP + transport, the specific role of these residues in DAT function remains unclear. Subsequent mutation of rDAT/ Y533 to phenylalanine, the corresponding residue in human DAT (hDAT), increased the velocity of MPP + uptake, suggesting that F533 in hDAT was important for MPP + uptake (Mitsuhata et al. 1998). Using the available mutation data from DAT, Ravna and Edvardsen (2001)

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

“…To examine the distance between residues identified at the entrance to the pore, we took advantage of the available Cys single mutants and generated Cys double mutants in the C109A background (Table 1). Our laboratory has previously observed that hSERT F556C is sensitive to inactivation by MTSET (9). In the present study, F556C as well as (Fig.…”

“…In this study, a homology model of SERT based on the crystal structure of a member of the neurotransmitter sodium symporter family was used to obtain insights into the extracellular entrance to the substrate permeation pathway (9,10). Our model suggests that the entrance to the pore is lined by TMHs I, III, VI, X, and XI as well as EL4 (Fig.…”

“…Surface expression binding experiments were performed in 24-well plates (1 ϫ 10 5 cells/well) precoated with poly-D-lysine as described previously (9). The day after plating, cells were washed once with KRH buffer.…”

Recent advances in the understanding of the interaction of antidepressant drugs with serotonin and norepinephrine transporters

“…A widely used approach has been the substituted-cysteine accessibility method (SCAM), where cysteines are introduced in different positions and selectively derivatized with thiol reactive compounds, to provide information on protein structure. [98][99][100] Several SCAM studies on SERT [101][102][103][104][105][106][107][108][109] and NET 110 have verified the overall 12 TMD topology as well as identified a number of amino acid positions as potential candidates for binding site residues.…”