Determination of External Loop Topology in the Serotonin Transporter by Site-directed Chemical Labeling

Abstract

The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEAbiotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and ␥-aminobutyric acid transporters. The serotonin transporter (SERT)1 is a member of the NaCldependent transporter family, a large group of homologous proteins that cotransport their substrates together with Na ϩ and Cl Ϫ ions (1-5). The substrates for this transporter family include neurotransmitters such as serotonin (5-HT), norepinephrine, dopamine, ␥-aminobutyric acid (GABA), and glycine as well as other substances such as proline, creatine, betaine, and taurine (6 -16). The biogenic amine transporters, SERT, the dopamine transporter, and the norepinephrine transporter (NET) are important in vivo targets for antidepressant and psychostimulant drugs (17-21).As a result of the high level of sequence conservation within the NaCl-dependent transporter family, hydropathy profiles of the various transporters are almost identical. These profiles have been interpreted as evidence for 12 transmembrane domains with the NH 2 -and COOH-terminal domains in the cytoplasm (17,22). Understanding the topological organization of these transporters is essential for any thorough analysis of their structure. Some features of the originally proposed topology have been confirmed experimentally, but others are in dispute. Each member of the family has one or more potential sites for glycosylation in the second predicted external hydrophilic loop (EL2). These glycosylation sites have been removed in SERT (23), NET (24), the GABA transporter GAT-1 (25), and the glycine transporter GLYT-1 (26). In each case, the el...

Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes , Determined by Site-Directed Fluorescence Labeling

“…As a result, we felt it necessary to examine topology in a way that preserved the normal structure of our target. Although several methods provide topological information in the context of an intact protein (7,17,29,37,38), we chose to capitalize on the targeted insertion of cysteine into an otherwise cysteine-less host. In earlier studies using this approach (18,23), topology was inferred by asking if the labeling of a target cysteine by a membrane-permeable probe could be blocked by an impermeable thiol-specific agent.…”

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

“…After 2 h of induction at 30°C (final optical density ϭ 0.6), 4 ml of the culture were employed for the biotinylation tests. Cell surface proteins were labeled with the membrane-impermeant biotinylation reagent, NHS-SS-biotin (Pierce), as described previously (22). Cells were incubated for 20 min at 4°C with NHS-SS-biotin in alkaline medium, pH 8.5, the excess reagent was quenched with glycine, and the cells were dissolved in Triton X-100 and SDS.…”

“…This reagent is particularly useful because of its high accessibility to small external loops, and it has been employed recently to characterize the external loop topology in the serotonin transporter (22). Cells were treated with this reagent and were then solubilized in detergent.…”

Analysis of Transmembrane Domain 2 of Rat Serotonin Transporter by Cysteine Scanning Mutagenesis

“…Studies using site-directed chemical labeling of SERT have confirmed this topology (11,12). However, the boundaries between TM and loop domains remain largely unknown.…”