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

Abstract

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...

Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

“…Another and more likely explanation is that Thr-258 and Ser-259 only might be exposed in certain conformation during the transport cycle and/or to allow phosphorylation in the milieu of the lipid-mobile phase of mammalian plasma membrane; hence, the LeuT structure only represents a single snapshot of a highly dynamic protein in which both the putative extracellular and intracellular gates are closed. Interestingly, a recent study using cysteinescanning mutagenesis showed that in SERT, the region between TMD4 and TMD5 encompassing Thr-276 and Ser-277 (Thr-258 and Ser-259 in NET) is accessible from the cytoplasmic side to cysteine-reactive reagents suggesting that ICL2 is much longer than the initially predicted sequence, at least in certain conformations (59).…”

Mechanism for alternating access in neurotransmitter transporters

“…43. Binding to membranes was measured before and after incubation with MTSEA or MTSES (19), and typical results are shown in Fig. S1.…”

“…19. Each mutant was tested for transport activity and sensitivity to MTS reagents by transfecting HeLa cells with plasmids bearing each mutant cDNA under control of a T7 promoter and infecting with vTF7-3, a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase (42).…”

“…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). These reactive positions were proposed to contribute to the cytoplasmic substrate permeation pathway because their reactivity was modulated by substrates (serotonin, Na ϩ and Cl Ϫ ) and an inhibitor (cocaine) in a manner consistent with the effects of these ligands in the transport cycle (20).…”

Identification of a chloride ion binding site in Na + /Cl − -dependent transporters

“…All mutants were sequenced to verify the accuracy of the procedure. Transport measurements were performed as described (37). Briefly, HeLa cells transfected with SERT mutant DNA using the vaccinia T7 system (38) were grown in 24-or 48-well plates, rinsed into medium containing [ 3 H]5-HT and the indicated Cl Ϫ concentration, incubated 10 min at 25°C, rinsed again, and counted.…”