The Cytoplasmic Substrate Permeation Pathway of Serotonin Transporter

Abstract

Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na ؉ and Cl ؊ , which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation. Serotonin transporter (SERT)2 is a polytopic membrane protein responsible for reuptake of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) following its release (1). It is a member of the NSS or SLC6 family of sodium-coupled neurotransmitter and amino acid transporters, which also includes transporters for dopamine, norepinephrine, ␥-aminobutyric acid, and glycine (2). SERT is a target for antidepressant drugs such as fluoxetine (Prozac) and for psychostimulants such as cocaine and 3,4-methylenedioxymethamphetamine (also known as ecstasy) (3, 4). Antidepressants, cocaine and the high affinity cocaine analog 2␤-carbomethoxy-3␤-(4-iodophenyl)tropane (␤-CIT, or RTI-55) are competitive inhibitors of SERT-mediated 5-HT uptake and are likely to bind from the extracellular medium close to the 5-HT binding site on SERT (5-7).SERT couples the transport of one 5-HT molecule into the cell together with one Na ϩ and one Cl Ϫ ion and in exchange for one K ϩ ion in each catalytic cycle (8 -10). According to the alternating access model, a transporter contains a central binding site for substrates that is exposed alternately to either side of the membrane through conformational changes (11, 12). As a corollary to the alternating access model, when a transporter couples the transmembrane movement of more than one substrate, the process requires, for maximum efficiency, that the conformational change occurs only when all substrates that are transported in the same direction (symport) are bound (13). When substrates are transported in opposite directions (antiport) efficient coupling requires that the conformational change occurs only when substrate is bound and not when the bind...

Mechanism for alternating access in neurotransmitter transporters

“…2, Lower). Cocaine is expected to stabilize the extracellular state of SERT, in which the cytoplasmic pathway is closed (20); consistent with this expectation, cocaine decreased reactivity for the majority (11 of 13) of the most reactive mutants by 2-to 28-fold ( Fig. 2 and Fig.…”

“…To better define the cytoplasmic pathway, we have examined the cytoplasmic accessibility of residues in helices 6b, 8 and 1a in SERT in addition to TM5 (20,21). Our results suggest that the cytoplasmic vestibule is formed by the topologically inverted helices corresponding to those that form the extracellular vestibule, which is consistent with the internal symmetry of the protein.…”

“…In these membrane preparations, both cytoplasmic and extracellular faces are accessible to ligands and MTS reagents (20). Specifically, binding of the cocaine analogue 2␤-2-carbomethoxy-3-(4-iodophenyl)tropane (␤-CIT), was inhibited by MTS reagents that modified TM5 residues in the cytoplasmic permeation pathway (20).…”

“…In these membrane preparations, both cytoplasmic and extracellular faces are accessible to ligands and MTS reagents (20). Specifically, binding of the cocaine analogue 2␤-2-carbomethoxy-3-(4-iodophenyl)tropane (␤-CIT), was inhibited by MTS reagents that modified TM5 residues in the cytoplasmic permeation pathway (20). This inactivation is attributed to conformational restrictions, imposed by adding bulk and charge within the cytoplasmic pathway, that prevent SERT from assuming the extracellular-facing state to which cocaine and its analogues, such as ␤-CIT, bind.…”

“…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). Moreover, ibogaine, an hallucinogenic alkaloid proposed to bind preferentially to the cytoplasm-facing state of SERT, induced a concerted conformational change leading to increased accessibility of the reactive positions in TM5 and decreased reactivity of residues in the extracellular permeation pathway (21).…”