Hanhe Liu

Background: Albizia julibrissin Durazz. is one of the most popular herbs used for depression treatment, but the molecular basis for its mechanism of action has not been fully addressed. Previously, we isolated and identified two lignan glycoside derivatives that were shown to noncompetitively inhibit serotonin transporter (SERT) activity but with a relatively low inhibitory potency compared with those of conventional antidepressants. Methods: We characterized the pharmacological profile of the parental compound of these previously isolated lignan glycosides, (-)-syringaresinol (SYR), in inhibiting SERT by using biochemical, pharmacological, and behavioral approaches. Results: SYR, as a potent inhibitor, decreases SERT V_max but with little change in K_m for its fluorescent substrate. SYR was shown to block the conformational conversion essential for substrate transport by stabilizing SERT in an outward-open and inward-closed conformation. In addition, our molecular docking and biochemical validation demonstrated that SYR binds to an allosteric site in SERT and noncompetitively inhibits SERT transport and binding activity. Furthermore, administration of SYR was indicated to exert an antidepressant-like activity and to effectively attenuate chronic unpredictable mild stress (CUMS)-induced abnormalities in behaviors and synaptic protein expression in depressive animal models. Conclusions: This study not only provides molecular insights into the mechanism of action of A. julibrissin in the treatment of depression, but also opens up the possibility of development of a novel class of allosteric site-targeted therapeutic agents with an underlying mechanism of action different from that of conventional antidepressants.

All resolved high-resolution structures of the transporters in the neurotransmitter sodium symporter (NSS) family revealed that the NSS members share common structural and mechanistic features for substrate and ion binding and transport. However, a recently reported bacterial orthologue of the human serotonin transporter (hSERT), TuriSERT, possesses a structural characteristic specific for amino acid substrate binding but does transport a biogenic amine. The unique structural feature of TuriSERT requires a novel configuration for coordinating its substrate and ions. In the present study, we characterized TuriSERT expressed in Escherichia coli cells with a fluorescent substrate by biochemical, structural, and pharmacological approaches. Substrate transport by TuriSERT requires Na⁺ but not Cl⁻. Replacement of Asp262 by asparagine renders TuriSERT Cl⁻-dependent. Substitutions of the corresponding Na1 residues did not alter Na⁺ dependence on substrate transport, whereas the mutation of a Na2 site residue led to a loss of transport activity, suggesting that Na⁺ binds only to the Na2 site in TuriSERT. In addition, substitutions of several residues essential for recognizing 5-hydroxytryptamine (5-HT) in hSERT had little effect on 5-HT displacement potency in transport assay for TuriSERT. In contrast, mutations of the residues that are proposed to coordinate with 5-HT in our docking model dramatically reduced 5-HT displacement. Furthermore, our results indicated that all tested antidepressants showed a weak inhibitory effect on TuriSERT. The present study demonstrated the existence of a unique substrate binding site and 1:1 stoichiometry of sodium–substrate binding in TuriSERT, a novel structural finding for the NSS transporters.

Glycine transporter 1 (GlyT1) is responsible for the reuptake of glycine, which regulates glutamate signaling as a co-agonist with N-methyl-D-aspartic acid (NMDA) receptors in the excitatory synapse and has been proposed to be a potential target in the development of therapies for a broad range of disorders of the central nervous system. Despite significant progress in characterizing structure and transport mechanism of the transporter, the regulation of transport function through oligomerization remains to be understood. In the present work, association of two forms of GlyT1 into dimers and higher order oligomers was detected by coimmunoprecipitation. To investigate functional properties of dimers of a GlyT1 cysteine mutant L288C, we performed oxidative cross-linking of the positioned cysteine residues in extracellular loop 3 (EL3) near the extracellular end of TM6. By analyzing the effect of copper phenanthroline (CuP)-induced dimerization on transport function, cross-linking of L288C was found to inhibit transport activity. In addition, an intramolecular ion pair Lys286-Glu289 was revealed to be critical for stabilizing EL3 in a conformation that modulates CuP-induced dimerization and transport function of the GlyT1 L288C mutant. Furthermore, the influence of transporter conformation on GlyT1 L288C dimerization was investigated. The substrate glycine, in the presence of both Na⁺ and Cl⁻, significantly reduced oxidative cross-linking, suggesting a large-scale rotation of the bundle domain during substrate transport impairs interfacial interactions between L288C protomers. The present study provides new insights into structural and functional elements regulating GlyT1 transport activity through its dimerization or oligomerization.

Albizia julibrissin Durazz. is one of the most common herbs used for depression and anxiety treatment, but its molecular basis and mechanism of action as an antidepressant or anxiolytic drug are not understood. In this study, we separated and identified two lignan glycosides that inhibit serotonin transporter (SERT) noncompetitively by decreasing V_max with little change in K_m for its fluorescence substrate. In addition, treatment with lignan glycosides did not alter total and cell surface expression levels of the transporter protein. The two compounds decreased the accessibility of a cysteine residue placed in the extracellular substrate permeation pathway by inducing a conformational shift toward an outward-closed state of SERT. These results are consistent with molecular docking for the association of the lignan glycosides to the allosteric site in SERT. The present work supports the proposal that these compounds act on SERT by a novel underlying mechanism of action different from that of conventional antidepressant drugs.