The serotonin type 3A (5-HT 3A ) receptor is a homopentameric cation-selective member of the pentameric ligandgated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains: extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Previously, we have demonstrated that the 5-HT 3A -ICD is required for the interaction between 5-HT 3A and the chaperone protein resistance to inhibitors of choline esterase (RIC-3). Additionally, we have shown that 5-HT 3A -ICD fused to maltose-binding protein (MBP) directly interacts with RIC-3, without the involvement of other protein(s). To elucidate the molecular determinants of this interaction, we developed different MBP-fused 5-HT 3A -ICD constructs by deleting large segments of its amino acid sequence. We expressed seven engineered ICDs in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction between MBP-5HT 3A -ICD constructs and RIC-3 was investigated. In summary, we identify a 24-amino-acid-long segment of the 5-HT 3A -ICD as a molecular determinant for the interaction between the 5-HT 3A -ICD and RIC-3.

Serotonin type 3 receptors (5-HT3Rs) are cation-conducting pentameric ligand-gated ion channels and members of the Cys-loop superfamily in eukaryotes. 5-HT3Rs are found in the peripheral and central nervous system, and they are targets for drugs used to treat anxiety, drug dependence, and schizophrenia, as well as chemotherapy-induced and postoperative nausea and emesis. Decades of research of Cys-loop receptors have identified motifs in both the extracellular and transmembrane domains that mediate pentameric assembly. Those efforts have largely ignored the most diverse domain of these channels, the intracellular domain (ICD). Here we identify molecular determinants within the ICD of serotonin type 3A (5-HT3A) subunits for pentameric assembly by first identifying the segments contributing to pentamerization using deletion constructs of, and finally by making defined amino acid substitutions within, an isolated soluble ICD. Our work provides direct experimental evidence for the contribution of three intracellular arginines, previously implicated in governing the low conductance of 5-HT3ARs, in structural features such as pentameric assembly.

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT) receptors.

Bupropion, a Food and Drug Administration-approved antidepressant and smoking cessation aid, blocks dopamine and norepinephrine reuptake transporters and noncompetitively inhibits nicotinic acetylcholine and serotonin (5-HT) type 3A receptors (5-HT 3A Rs). 5-HT 3 receptors are pentameric ligandgated ion channels that regulate synaptic activity in the central and peripheral nervous system, presynaptically and postsynaptically. In the present study, we examined and compared the effect of bupropion and its active metabolite hydroxybupropion on mouse homomeric 5-HT 3A and heteromeric 5-HT 3AB receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp experiments. Coapplication of bupropion or hydroxybupropion with 5-HT dose dependently inhibited 5-HT-induced currents in heteromeric 5-HT type 3AB receptors (5-HT 3AB Rs) (IC 50 5 840 and 526 mM, respectively). The corresponding IC 50 s for bupropion and hydroxybupropion for homomeric 5-HT 3A Rs were 10-and 5-fold lower, respectively (87 and 113 mM). The inhibition of 5-HT 3A Rs and 5-HT 3AB Rs was non-use dependent and voltage independent, suggesting bupropion is not an open channel blocker. The inhibition by bupropion was reversible and time-dependent. Of note, preincubation with a low concentration of bupropion that mimics therapeutic drug conditions inhibits 5-HT-induced currents in 5-HT 3A and 5-HT 3AB receptors considerably. In summary, we demonstrate that bupropion inhibits heteromeric 5-HT 3AB Rs as well as homomeric 5-HT 3A Rs. This inhibition occurs at clinically relevant concentrations and may contribute to bupropion's clinical effects. SIGNIFICANCE STATEMENT Clinical studies indicate that antagonizing serotonin (5-HT) type 3AB (5-HT 3AB) receptors in brain areas involved in mood regulation is successful in treating mood and anxiety disorders. Previously, bupropion was shown to be an antagonist at homopentameric 5-HT type 3A receptors. The present work provides novel insights into the pharmacological effects that bupropion exerts on heteromeric 5-HT 3AB receptors, in particular when constantly present at low, clinically attainable concentrations. The results advance the knowledge on the clinical effects of bupropion as an antidepressant.