Functional Role of Critical Stripe Residues in Transmembrane Span 7 of the Serotonin Transporter

Abstract

Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na ؉ dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted ␣-helix, formed part of a water-filled pore for Na ؉ . We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter's three-dimensional structure.

The Aqueous Accessibility in the External Half of Transmembrane Domain I of the GABA Transporter GAT-1 Is Modulated by Its Ligands

“…1) and the same conclusion was reached in SERT (21). In this gene family cysteine substitution was quite well tolerated in other TMDs and loops (17,28,32). It appears therefore that the heavily impacted external half of TMD I fulfils an important role in neurotransmitter transport in line with previous observations on its importance in the interaction with the neurotransmitter (18,19).…”

A Lithium-induced Conformational Change in Serotonin Transporter Alters Cocaine Binding, Ion Conductance, and Reactivity of Cys-109

“…Together with data from the F380Y mutant of SERT (24), the present data suggest that for SERT also, Na ϩ induces a conformational change or stabilizes the native conformation of SERT to oppose the conformational change induced by Li ϩ . In previous studies Li ϩ has been used as an inert replacement for Na ϩ (4,13,14).…”

“…2 We consider it more likely that Na ϩ and Li ϩ , acting at different sites, both induce conformational changes in the transporter and that these changes are mutually exclusive. Recent results with the F380Y mutant of SERT indicates the opposing effects of Na ϩ and Li ϩ on the reactivity of Cys-109 (24). Another indication of this phenomenon is that Na ϩ and Li ϩ had opposite effects on ␤-CIT binding to SERT (Fig.…”

The Chicken Serotonin Transporter Discriminates between Serotonin-selective Reuptake Inhibitors

“…The important role of TM1 was supported by the suggestion of G100 as a structural pivot providing flexibility during substrate translocation (33). In TM7, several residues are important for uptake and Na ϩ dependence, indicating that TM7 is involved in binding pocket formation or substrate translocation (34). A cysteine scanning of residues in TM3 indicated that Ile 172 and Tyr 176 are associated with a 5-HT and cocaine binding pocket (35).…”