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Serotonin or 5-hydroxytryptamine receptors type 3 (5-HT3) belong to the family of pentameric ligand-gated ion channels (pLGICs), which also includes other neurotransmitter-gated ion channels such as nicotinic acetylcholine receptors (nAChRs). pLGICs have been long-standing therapeutic targets for psychiatric disorders such as anxiety, schizophrenia, and addiction, and neurological diseases like Alzheimer’s and Parkinson’s disease. Due to structural conservation and significant sequence similarities of pLGICs’ extracellular and transmembrane domains across the more than 40 subunits found in humans, clinical trials for drug candidates targeting these two domains have been largely hampered by undesired effects mediated by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 directly interacts with the ICD of 5-HT3A subunits. Using a sequential deletion approach, we identified the L1-MX segment of the ICD fused to maltose-binding protein as sufficient for the interaction. For the present study, synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay identified positions W347, R349, and L353 as critical for binding to RIC-3. In complementary studies with full-length 5-HT3A subunits, the identified Ala substitutions reduced the modulation of functional surface expression by co-expression of RIC-3. Additionally, we found and characterized a duplication of the binding motif at the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary we identify two binding sites for RIC-3 with a shared duplicated motif in 5-HT3A subunits, one in the MX-helix and one at the MAM4-helix transition.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.HighlightsCell free expression of PCFT without any lipids or detergents resulted in quantitative precipitation of in-situ synthesized PCFT.Additives for expression of PCFT in the soluble fraction were identified.

Serotonin type 3A receptors (5-HT 3A Rs) are pentameric ligand-gated ion channels, pharmacologically targeted for the treatment of severe nausea and vomiting. The intracellular domain of 5-HT 3A Rs has been shown to be a crucial determinant for limiting conductance. Structurally, it consists of a short L1-loop following the third transmembrane segment M3, a short α-helical MX-segment, a large unstructured L2-loop, and the membrane-associated MA-helix that continues into the last transmembrane segment M4. During gating, conformational changes occur in all three domains. Extracellular and transmembrane domains rotate counterclockwise and clockwise, respectively. Within the intracellular domain the MA-M4 helix breaks at Gly430, leading to pore widening above and below the helix break, and the MX-helix moves outward and upward towards the membrane. At their bottom, the MA-helices come into close apposition to shape a narrow hydrophobic constriction below the cytosolic channel chalice that, in its upper section, is framed by lateral windows through which the L1-loops thread. In the present study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of the bottom segments of the MA-helices. Repeated agonist application or oxidation induced a current run down for channels with Cys pairs at I409C/R410C. For Cys pairs at L402C/L403C these conditions did not lead to altered currents. On the contrary, cross-linked subunits for both L402C/L403C and I409C/R410C were observed for both conditions using gel electrophoresis. Our results indicate that the proximity and orientation for Cys in both pairs is conducive for disulfide bond formation. While conformational changes associated with gating promote cross-linking for I409C/R410C that in turn inhibits gating, cross-linking of L402C/L403C is functionally silent. We infer that conformational changes associated with gating are more pronounced for the upper I409C/R410C pair in close proximity to the lateral windows as compared to the L402C/L403C pair at the apex of the inverted pentagonal pyramid contributed by the MA-helices. Statement of Significance The intracellular domain (ICD) of pentameric ligand-gated ion channels (pLGICs) is the most diverse domain within receptors of the Cys-loop superfamily. Despite being the least understood domain of pLGICs, we know that it influences channel function in multiple ways and shapes the cytosolic exit pathway of the channel. X-ray and cryo-EM structures have captured the structured segments of the ICD of 5-HT 3A receptors in different conformational states with lower resolution of the ICD as compared to the other domains. Here, we provide experimentally-derived evidence for the importance of the mobility of the MA-helices and functionally confirm ion-conduction through lateral portals as opposed to a vertical pathway for 5-HT 3A receptors.

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. However, the specific ion conduction pathway through this domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a “closed vestibule”, with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In the present study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. While conformational changes associated with gating promote crosslinking for I409C/R410C, which in turn decreases channel currents, crosslinking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely.SignificanceThe intracellular domain (ICD) of pentameric ligand-gated ion channels (pLGICs) is the most diverse domain within receptors of the Cys-loop superfamily. Despite being the least understood domain of pLGICs, its impact on ion-channel function and contribution to the cytosolic exit pathway of the channel have been investigated. X-ray and cryo-EM structures have captured the structured segments of the ICD of 5-HT3A receptors in different conformational states with lower resolution of the ICD as compared to the other domains. Here, we provide experimentally derived evidence for the importance of the differential mobility of the cytosolic segment of the MA-helices, which supports the existence of lateral portals as opposed to a vertical pathway for 5-HT3A receptors.

Bupropion, a FDA-approved antidepressant and smoking cessation aid, blocks dopamine and norepinephrine reuptake transporters and non-competitively inhibits nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3) receptors. 5-HT3 receptors are pentameric ligand-gated ion channels that regulate synaptic activity in the central and peripheral nervous system pre- and postsynaptically. In the present study, we examined and compared the effect of bupropion and its active metabolite hydroxybupropion on homomeric 5-HT3A and heteromeric mouse 5-HT3AB receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp experiments. Co-application of bupropion or hydroxybupropion with 5-HT dose-dependently inhibited 5-HT-induced currents in 5-HT3ABRs (IC50 = 866 μM and 505 μM, respectively) but potentiated 5-HT-induced currents at low (30-50 μM) concentrations. The corresponding IC50s for bupropion and hydroxybupropion with 5-HT3AR were 10- and 5-fold lower, respectively (87 μM and 113 μM), and no potentiation was observed. The inhibition of 5-HT3AR and 5-HT3ABR was non-use dependent and voltage-independent, indicating bupropion is not an open channel blocker. The inhibition by bupropion was reversible and time-dependent. Of note, pre-incubation with a low concentration of bupropion that mimics therapeutic drug conditions significantly inhibited 5-HT induced currents in 5-HT3A and even more so 5-HT3AB receptors. In summary, our results indicate that bupropion inhibits 5-HT3ABR, as well as homomeric receptors, and that this inhibition takes place at clinically-relevant concentrations. Inhibition of 5-HT3 receptors by bupropion may contribute to its desired and/or undesired clinical effects.Significance Statement5-HT3AB receptors are found in brain areas involved in mood regulation. Clinical studies indicate that antagonizing these receptors was successful in treating mood and anxiety disorders. Some currently clinically available antidepressants and antipsychotics act as antagonists of 5-HT3 receptors. Previously, bupropion was shown to be an antagonist at homopentameric 5-HT3A receptors. The present work provides novel insights into the pharmacological effects bupropion exerts on heteromeric 5-HT3AB receptors. The results advance the knowledge on the clinical effect of bupropion as an antidepressant.

The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated 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 also demonstrated that 5-HT3A-ICD is required and sufficient for the interaction between 5-HT3A and RIC-3. Additionally, we have shown that 5-HT3A-ICD fused to maltose binding protein (MBP) directly interacts with the chaperone protein resistance to inhibitors of choline esterase (RIC-3), without the involvement of other protein(s). To elucidate the molecular determinants of this interaction we developed different MBP-fused 5-HT3A-ICD constructs by deletion of large portions of its amino acid sequence. We have expressed seven mutants in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction of MBP-5HT3A-ICD constructs and RIC-3 is investigated. Furthermore, we co-expressed 5-HT3A and 5-HT3AB, a heteromeric form of 5-HT3Rs, with RIC-3 in Xenopus oocytes to compare their interaction with RIC-3 in-vivo by two electrode voltage clamp (TEVC) recordings. Full-length 5-HT3A-and 5-HT3AB mediated currents are significantly reduced when RIC-3 is co-expressed in either condition. In summary, we identify a 24-amino acid long segment of the 5-HT3A-ICD as a molecular determinant for the interaction between the 5-HT3A-ICD and RIC-3.Statement of SignificanceThe chaperone protein RIC-3 is known to modulate the functional surface expression of cation-conducting pentameric ligand-gated ion channels. Previously we have demonstrated that the intracellular domain of serotonin channels mediates this effect. Here we provide experimental evidence for a 24-amino acid long segment within the 115-amino acid long intracellular domain as a determinant for RIC-3 interaction. Recently it was found experimentally that the identified segment contains an alpha helix that has been observed or predicted to be present in other cation-conducting channels. The present work provides novel insights into protein-protein interactions that are likely also relevant for other cation-conducting members of this large ion channel family that includes nACh and 5-HT3 receptors.