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Background and Purpose The α4β2 nicotinic ACh receptor (nAChR), a subtype of the ligand‐gated ion channel, is abundantly expressed in the brain and is implicated in several neurological disorders. The endocytosis of nAChRs plays important roles in the pathogenesis of neurological diseases, but the underlying molecular mechanisms remain poorly understood. Experimental Approach Loss‐of‐function approaches and mutants of α4β2 nAChRs that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signalling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChRs was determined and crosschecked using an ELISA and radioligand assay. Key Results Endocytosis of α4β2 nAChRs occurred through clathrin‐mediated endocytosis in a dynamin‐dependent manner. 14‐3‐3η‐dependent Src‐mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β‐arrestin1 and GPCR kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homologue‐mediated ubiquitination and subsequent down‐regulation of α4β2 nAChRs. Conclusions and Implications α4β2 nAChR, an ionophore receptor, employs the metabotropic signalling pathway required for endocytosis, which leads to ubiquitination and down‐regulation. Further, GRK2 and β‐arrestin1, usually associated with GPCR signalling, are involved in the endocytosis of α4β2 nAChRs via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.

Tetrazoles were designed and synthesized as potential inhibitors of triple monoamine neurotransmitters (dopamine, norepinephrine, serotonin) reuptake based on the functional and docking simulation of compound 6 which were performed in a previous study. The compound structure consisted of a tetrazole-linker (n)-piperidine/piperazine-spacer (m)-phenyl ring, with tetrazole attached to two phenyl rings (R1 and R2). Altering the carbon number in the linker (n) from 3 to 4 and in the spacer (m) from 0 to 1 increased the potency of serotonin reuptake inhibition. Depending on the nature of piperidine/piperazine, the substituents at R1 and R2 exerted various effects in determining their inhibitory effects on monoamine reuptake. Docking study showed that the selectivity of tetrazole for different transporters was determined based on multiple interactions with various residues on transporters, including hydrophobic residues on transmembrane domains 1, 3, 6, and 8. Co-expression of dopamine transporter, which lowers dopamine concentration in the biophase by uptaking dopamine into the cells, inhibited the dopamine-induced endoctytosis of dopamine D2 receptor. When tested for compound 40 and 56 , compound 40 which has more potent inhibitory activity on dopamine reuptake more strongly disinhibited the inhibitory activity of dopamine transporter on the endocytosis of dopamine D2 receptor. Overall, we identified candidate inhibitors of triple monoamine neurotransmitter reuptake and provided a theoretical background for identifying such neurotransmitter modifiers for developing novel therapeutic agents of various neuropsychiatric disorders.

Numerous psychotropic and addictive substances possess structural features similar to those of -phenethylamine (-PEA). In this study, we selected 29 -PEA derivatives and determined their structure-activity relationship (SAR) to their ability to inhibit dopamine (DA) reuptake; conducted docking simulation for two selected compounds; and identified their potential functionals. The compounds were subdivided into arylethylamines, 2-(alkyl amino)-1-arylalkan-1-one derivatives and alkyl 2-phenyl-2-(piperidin-2yl)acetate derivatives. An aromatic group, alkyl group, and alkylamine derivative were attached to the arylethylamine and 2-(alkyl amino)-1-arylalkan-1-one derivatives. The inhibitory effect of the compounds on dopamine reuptake increased in the order of the compounds substituted with phenyl, thiophenyl, and substituted phenyl groups in the aromatic position; compounds with longer alkyl groups and smaller ring-sized compounds at the alkylamine position showed stronger inhibitory activities. Docking simulation conducted for two compounds, 9 and 28, showed that the (S)-form of compound 9 was more stable than the (R)-form, with a good fit into the binding site covered by helices 1, 3, and 6 of human dopamine transporter (hDAT). In contrast, the (R, S)-configuration of compound 28 was more stable than that of other isomers and was firmly placed in the binding pocket of DAT bound to DA. DAinduced endocytosis of dopamine D2 receptors was inhibited when they were co-expressed with DAT, which lowered extracellular DA levels, and uninhibited when they were pretreated with compound 9 or 28. In summary, this study revealed critical structural features responsible for the inhibition of DA reuptake and the functional role of DA reuptake inhibitors in regulating D2 receptor function.