The Role of External Loop Regions in Serotonin Transport

Abstract

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na ؉ . In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter. The serotonin transporter (SERT)1 is responsible for the accumulation of serotonin (5-hydroxytryptamine, 5-HT) by neurons, platelets, and other cells (1-4). It is a member of a large family of carriers that couple the uphill movement of neurotransmitters and other metabolites with the downhill flux of Na ϩ and Cl Ϫ (5). The strong homology within this family suggests that the mechanism of transport is similar and that structural elements conserved within the family perform similar functions in each transporter. A prominent feature in each of the primary sequences is the presence of 12 regions rich in hydrophobic amino acids, linked by hydrophilic regions of variable length (6, 7). The hydrophobic regions were originally modeled as membrane-spanning domains linked by alternating external and internal loops (6, 7). One of these loops, which links putative transmembrane domains 3 and 4 (TM3 and TM4), is much larger than the rest and contains consensus sites for N-linked glycosylation (8) and an intramolecular disulfide (9) in all family members.The original topological model for these NaCl-coupled transporters was challenged in studies using mutants of the ␥-aminobutyric acid (GABA) and glycine transporters (GAT-1 and GLYT-1) (10, 11). These studies suggested that the predicted first external loop (EL1) was not exposed on the cell exterior and that the first internal loop (IL1) was actually extracellular. One drawback of these studies was that the conclusions about EL1 and IL1 topology were drawn from the prope...

Substrate-induced Conformational Changes of Extracellular Loop 1 in the Glycine Transporter GLYT2

“…This process turns the outward-facing form of the protein, in which residue 223 is externally exposed, into the inward-facing form, where this residue is occluded from the external medium. Although a relevant role in substrate or inhibitor binding has been reported for hydrophilic loops in the ␥-aminobutyric acid transporters (54), it has also been recently suggested that the complementary functions of binding and conformational changes in the transport cycle are subserved by the transmembrane and loop domains, respectively (55). Our results may also reinforce this hypothesis.…”

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

“…Evidence from external loop chimeras suggests that these loops are important for the conformational changes that follow binding (22). It is conceivable that the structural basis for our observations lies in the conformational changes in the first external loop, which contains Cys-109, that accompany ligand and ion binding to SERT.…”

Localization of Cocaine Analog [125I]RTI 82 Irreversible Binding to Transmembrane Domain 6 of the Dopamine Transporter

“…We cannot at present exclude the possibility that incorporation of the ligand occurs in loop structures but will focus on the transmembrane spanning sequence as the likely site of attachment, because most currently available evidence indicates that determinants for uptake blocker binding in DAT and related transporters are present in TMs rather than in loops (21,22 (27), whereas increased cocaine affinity occurred at D313N (26). Most of the inhibitory mutations decreased cocaine affinity by only modest amounts (2-to 3-fold) that may be consistent with indirect effects on binding site structure rather than alteration of direct interaction sites, although mutation of multiple contact points may be required to reduce inhibitor affinity more significantly (28 (6).…”