Cysteine-Scanning Mutagenesis of the Fifth External Loop of Serotonin Transporter

Keller, Preston C.; Stephan, Megan M.; Glomska, Hanna; Rudnick, Gary

doi:10.1021/bi035971g

Cited by 29 publications

(30 citation statements)

References 36 publications

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“…These studies were carried out before the publication of the first LeuT structure (13) and according to the predicted topological models available at the time, the region studied was thought to represent extracellular loop 5, rather than the extracellular half of TM10. Many MTSET and MTSES accessible cysteine mutants were found, but this accessibility did not seem to be sensitive to the conformation of SERT (36), as opposed to our results with GAT-1. While we cannot exclude intrinsic mechanistic differences between GAT-1 and SERT, it is possible that the TM10 positions are also conformationally sensitive, but that the sensitivity is more difficult to detect in the SERT background.…”

Section: Discussioncontrasting

confidence: 99%

“…The SERT counterpart of GAT-1-D451C, E493C, retained very significant transport activity (36). Interestingly SERT, but not GAT-1, has an adjacent glutamate residue, Glu-494.…”

Section: Discussionmentioning

confidence: 98%

“…A cysteine scan of roughly the same residues shown here was performed in the serotonin transporter SERT (36). These studies were carried out before the publication of the first LeuT structure (13) and according to the predicted topological models available at the time, the region studied was thought to represent extracellular loop 5, rather than the extracellular half of TM10.…”

Section: Discussionmentioning

confidence: 99%

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An Acidic Amino Acid Transmembrane Helix 10 Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Formation of the Extracellular Gate of the γ-Aminobutyric Acid (GABA) Transporter GAT-1

Ben-Yona¹,

Kanner²

2012

Journal of Biological Chemistry

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Background: A conserved aspartate residue in the NSS family was suggested to participate in the formation of a "thin" extracellular gate. Results: Mutation of this residue increases the proportion of outward-facing transporters. Conclusion: This residue plays a crucial role in the gating mechanism of GAT-1. Significance: The new insights into GAT-1 gating may be relevant for other NSS transporters.

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Section: Discussioncontrasting

confidence: 99%

“…The SERT counterpart of GAT-1-D451C, E493C, retained very significant transport activity (36). Interestingly SERT, but not GAT-1, has an adjacent glutamate residue, Glu-494.…”

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

An Acidic Amino Acid Transmembrane Helix 10 Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Formation of the Extracellular Gate of the γ-Aminobutyric Acid (GABA) Transporter GAT-1

Ben-Yona¹,

Kanner²

2012

Journal of Biological Chemistry

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“…Consistent with the structure of LeuT, in the homologous serotonin transporter (SERT), TM10 was shown to be accessible to extracellular reagents (18). Conversely, in SERT, methanethiosulfonate (MTS) reagents applied from the cytoplasmic side of the membrane reacted rapidly with cysteine residues introduced into the cytoplasmic half of TM5, which corresponds to TM10 in the second repeat (19).…”

mentioning

confidence: 84%

Mechanism for alternating access in neurotransmitter transporters

Forrest

Zhang

Jacobs

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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362

483

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Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1-5 and TMs 6 -10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.alternating access mechanism ͉ neurotransmitter:sodium symporters ͉ serotonin ͉ structural modeling ͉ structural repeats

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“…Subsequent experiments using mutagenesis and chemical modification in intact cells and disrupted membranes have verified the overall topology (20,21). However, the extent of most of the loops remains unknown, with the exception of extracellular loops 4 (22) and 5 (23).…”

mentioning

confidence: 95%

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Zhang

Rudnick

2005

Journal of Biological Chemistry

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The serotonin transporter (SERT, 1 SLC6A4) is a presynaptic plasma membrane protein responsible for the reuptake of serotonin (5-hydroxytrypatamine, abbreviated 5-HT) after its release by neurons. It is a member of a large family of amine and amino acid transporters (SLC6, NSS) distributed throughout the prokaryotic and animal kingdoms. Together with norepinephrine and dopamine transporters (NET and DAT), SERT is part of a subgroup of transporters that mediate the transport of biogenic amine neurotransmitters by symport with Na ϩ and Cl Ϫ ions (1). SERT is of particular interest in neurobiology because it is the molecular target of several drugs of abuse and many therapeutic agents used to treat psychiatric disorders. Along with NET and DAT, SERT is inhibited by cocaine, and cocaine analogues bind to SERT with high affinity (2-4). Amphetamine and its derivatives, including 3,4-methylenedioxymethamphetamine (MDMA, also known as ecstasy), interact with SERT, DAT, and NET as substrates (5, 6). Inhibitors that prevent 5-HT reuptake into serotonergic neurons have been used to treat a variety of neuropsychiatric disorders, including affective disorder, anxiety disorder, obsessive-compulsive disorder, and autism (7-9). Furthermore, the incidence of some psychiatric disorders and the effectiveness of antidepressant drugs has been linked to polymorphisms in the promoter of the gene encoding SERT (10 -13).A naturally occurring mutation in human SERT, I425V, was found to be associated with obsessive-compulsive disorder and several other 5-HT-related disorders (14). Further investigation revealed that this mutation caused SERT to be in an activated state that is reached in the wild type through the action of cGMP-dependent protein kinase (15).Hydropathy analysis of SERT predicted 12 transmembrane (TM) domains connected by intracellular and extracellular loops (16,17). This analysis predicted that the second intracellular loop (IL2) was a short cytoplasmic loop containing nine residues connecting TM domains 4 and 5. Further analysis suggested that this loop contained consensus phosphorylation sites for cGMP-dependent protein kinase and protein kinase C (18). From the location of glycosylation sites in the second extracellular loop, the NH 2 and COOH termini were predicted to be cytoplasmic (19). Subsequent experiments using mutagenesis and chemical modification in intact cells and disrupted membranes have verified the overall topology (20, 21). However, the extent of most of the loops remains unknown, with the exception of extracellular loops 4 (22) and 5 (23).Previous studies of intracellular and extracellular loop residues by cysteine substitution have revealed changes in conformation of these loops in response to substrate and inhibitor binding (21,22,24,25). To further examine the structure of SERT IL2, we have studied IL2 by cysteine-scanning mutagenesis. The results reported here suggest that it is longer than predicted and contains an ␣-helical structure. EXPERIMENTAL PROCEDURESMutagenesis-Mutant transporters were gen...

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Cysteine-Scanning Mutagenesis of the Fifth External Loop of Serotonin Transporter

Cited by 29 publications

References 36 publications

An Acidic Amino Acid Transmembrane Helix 10 Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Formation of the Extracellular Gate of the γ-Aminobutyric Acid (GABA) Transporter GAT-1

An Acidic Amino Acid Transmembrane Helix 10 Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Formation of the Extracellular Gate of the γ-Aminobutyric Acid (GABA) Transporter GAT-1

Mechanism for alternating access in neurotransmitter transporters

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

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