The serotonin type 3A (5-HT3A) receptor is a pentameric ligand-gated ion channel (pLGIC) in central and peripheral neurons that conducts sodium and potassium ions upon serotonin binding. 5-HT3 receptors modulate neurotransmission and synaptic plasticity, influencing mood, sleep, appetite, and addiction. Disruptions in serotonin signaling are linked to central nervous system disorders, including schizophrenia, anxiety and depression. Clinically, these receptors are targeted by antagonists to treat chemotherapy-induced nausea and vomiting. The functional surface expression of these channels is regulated by the chaperone protein Resistant to Inhibitors of Cholinesterase 3 (RIC-3) that promotes plasma-membrane expression, maturation, and trafficking of 5-HT3A and nicotinic acetylcholine receptors. Our previous work identified a duplicated RIC-3 binding motif within the 5-HT3A intracellular domain (ICD). However, it was unclear whether this interaction reflected native conditions. Here, we used a recombinant 5-HT3A ICD peptide in peptide-resin pull-down assays to investigate RIC-3 Interactions in plasma membrane (PM) fractions from Xenopus oocytes, endoplasmic reticulum (ER) fractions from SH-SY5Y cells, and mouse brain tissue. Across all tested systems, the 5-HT3A ICD peptide specifically bound RIC-3. Furthermore, RIC-3 knockdown (RIC-3 KD) SH-SY5Y cells showed a marked reduction in peptide binding and decreased surface levels of nAChRα7 and 5-HT3A receptors. These results demonstrate RIC-3-5-HT3A ICD interaction in native cellular contexts and support a role for RIC-3 in regulating receptor surface expression and neuronal signaling.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric α7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of α7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT 3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the α7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of α7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic α7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of α7 oligomers and the biogenesis of α7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an α7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted α7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 °C, 45 °C, and 50 °C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish α7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARY:This work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of α7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels, such as nicotinic acetylcholine and serotonin type 3A receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pentameric ligandgated ion channels, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using the GLIC as a model, we used molecular docking to predict binding sites for bupropion. Bupropion was found to bind to several sites within the transmembrane domain, with the predominant site being localized to the interface between transmembrane segments M1 and M3 of two adjacent subunits. Residues W213, T214, and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within a 4 A & ring; distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the half-maximal inhibitory concentration to a 13-fold higher value compared to wildtype GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in the GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in g-aminobutyric acid type A receptors. Our data thus suggest that the T214 that lines a previously identified lipophilic binding pocket in GLIC and g-aminobutyric acid type A receptors is also a modulatory site for bupropion interaction with the GLIC.

ABSTRACT Bupropion is an atypical antidepressant and smoking cessation drug which causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. However, the binding sites and binding mechanisms of bupropion are still elusive. To further understand the inhibition of pLGICs by bupropion, in this work, using a prokaryotic homologue of pLGICs as a model, we examined the inhibitory potency of bupropion in Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated ion channel. Bupropion inhibited proton-induced currents in GLIC with an inhibitory potency of 14.9 ± 2.0 μM, comparable to clinically attainable concentrations previously shown to also modulate eukaryotic pLGICs. Using single amino acid substitutions in GLIC and two-electrode voltage-clamp recordings, we further determined a binding site for bupropion in the lower third of the first transmembrane segment M1 at residue T214. The sidechain of M1 T214 together with additional residues of M1 and also of M3 of the adjacent subunit have previously been shown to contribute to binding of other lipophilic molecules like allopregnanolone and pregnanolone. SIGNIFICANCE GLIC, Gloeobacter ligand-gated ion channel, has been extensively used as a model to identify and understand binding sites and mechanisms for cholesterol, neurosteroids, and anesthetics interacting with neurotransmitter-gated ion channels, such as, GABA A receptors and nicotinic acetylcholine receptors. Recently, increasing evidence has revealed that another neurotransmitter-gated ion channel, the serotonin type 3A receptor, binds to an atypical antidepressant, bupropion, at clinically relevant concentrations. Our work here proposes a binding site for bupropion in GLIC and suggests a new approach to characterize binding mechanisms of bupropion in other neurotransmitter-gated ion channels.

Serotonin type 3A (5-HT3A) receptors belong to the Cys-loop ligand-gated ion channel receptor superfamily. 5-HT3A receptors are found in high densities in certain brain regions, have an involvement with brain-gut signaling circuitry and other non-serotonergic synaptic activities, which make them effective and potential therapeutic targets for treatments of many conditions such as irritable bowel syndrome, chemotherapy-induced vomiting, inflammation, and psychiatric disorders. RIC-3, a transmembrane protein and endoplasmic reticulum resident chaperone, acts as a molecular chaperone of cation-conducting Cys-loop receptors, both serotonin type 3A (5-HT3A) and nicotinic acetylcholine receptors. RIC-3 is needed for efficient receptor folding, assembly and trafficking/functional surface expression. RIC-3 interacts directly with the intracellular domain of serotonin type 3A subunits (ICD). RIC-3 has two binding sites with a shared duplicated motif in 5HT3A subunits, one in the MX-helix and one in the MAM4-helix. Toward drug discovery based on protein-protein interactions, we here aimed to determine the effect of RIC-3 on the expression profile of serotonin type 3A receptors. For this study, 5-HT3A constructs were genetically engineered, and the functional surface expression was examined using two-electrode voltage clamp and immunoblot analysis/western blot. We found that when 5-HT3A channels were co-injected with RIC-3, RIC-3 inhibits 5-HT3A current amplitudes elicited by serotonin in Xenopus oocytes; Ala replacements at residues W347, R349, and L353 (MX) or residues W447, R449, and L454 (MAM4) on the ICD weakened the inhibitory effect of RIC-3 on the functional surface expression of the channels. We infer that in Xenopus oocytes, RIC-3 reduction of functional surface expression of 5HT3A receptors is mediated by binding to the two identified binding sites.

Serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors belong to the family of pentameric ligand-gated ion channels (pLGICs) that are therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities of pLGICs' extracellular and transmembrane domains, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A subunit intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In the present study, synthetic L1-MX-based peptides and Ala-scanning identify positions W347, R349, and L353 as critical for binding to RIC-3. Complementary studies using full-length 5-HT3A subunits confirm that the identified Ala substitutions reduce the RIC-3-mediated modulation of functional surface expression. Additionally, we find and characterize a duplication of the binding motif, DWLR…VLDR, present in both the MX-helix and 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 the binding motif for RIC-3 in 5-HT3A subunits at two locations in the ICD, one in the MX-helix and one at the MAM4-helix transition.

Pentameric ligand-gated ion channels (pLGICs) are cys-loop receptors important in transduction of electrical signals between neurons in the peripheral and central nervous system. Their significant role in neurotransmission has increased the research in neuropharmacology. Members of this protein family are found in mammals, but also in prokaryotes that can often incorporate additional domains whose roles are largely uncharacterized. pLGICs include nicotinic acetylcholinereceptors, gamma-amino butyric acid A receptors, glycine receptors and type 3 serotonin receptors (5HT3). These receptors are integral membrane protein complexes composed of five subunits that surround the central pore. Each subunit incorporates a large extracellular domain which contains the agonist-binding site, four transmembrane segments (TM1-4) which form the ion pore, a large intracellular domain (ICD) between TM3 and TM4, and a short extracellular C-terminal region. The ionotropic serotonin subtype-3 (5-HT3) receptor is of current interest as a therapeutic target in the treatment of alcohol abuse. Selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. To date only two applications have been fully realized in the clinic: the treatment of emesis and irritable-bowel syndrome. Competitive antagonists, called setrons, target 5HT3R, are used in the management of nausea and vomiting associated with radiation and chemotherapies in cancer patients. These drugs interact with off-target subunits of the receptor and consequently lead to many undesired effects including headache and drowsiness. In order to understand and characterize effective control of neurotransmitter-gated pLGICs, we have explored the interaction of 5HT3R with RIC-3 chaperone which mediates functional expression of the channels. In the present study, synthetic pLGIC peptides were used to probe and characterize the interaction with RIC-3 in native sources such as mouse brains and neuronal cell lines.

The α7 nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC). It is expressed in pivotal brain regions and plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. Their activation by the endogenous acetylcholine leads to signal transmission in the nervous system. Deficits in α7 function are associated with mental illnesses such as schizophrenia and degenerative conditions such as Alzheimer's disease. In the cholinergic anti-inflammatory pathway, binding of ligands to α7 nAChRs attenuates inflammation and may play a role in preventing the cytokine storm during SARS-CoV-2 infection. Undesired effects of nicotine binding to α7 nAChRs include proliferation, angiogenesis, and metastasis in lung cell carcinoma. In summary, the α7 nAChR has great potential as a therapeutic target. Current drug candidates for pGLICs in general bind to the extracellular domain (ECD) and transmembrane domain (TMD) of the receptor that are highly-homologous in this large super-family comprising more than 40 subunits in humans. Since the largest number of subunits belong to the nAChR family, interactions with off-target subunits, and consequently undesired effects are likely for ligands binding to the highly-conserved ECD or TMD. An understanding of the mechanism of action of α7 nAChR and of proteins affecting its activity may facilitate development of targeted therapies. Functional expression and properties of nAChRs have been shown to be affected by Resistance to Inhibitors of Cholinesterase-3 (RIC-3). Here, we explore the interaction of the intracellular domain (ICD) of α7 nAChR and cytoplasmic protein RIC-3 chaperon which mediates functional expression of the channel. Using synthetic peptides, the molecular interaction between α7 nAChRs and RIC-3 will be characterized, and the results will be substantiated using full-length α7 nAChRs in electrophysiological experiments after heterologous expression.

Serotonin or 5-hydroxytryptamine type 3A receptors (5-HT3A) belong to the pentameric ligand-gated ion channel super-family, which have been long-standing therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities in the extracellular and transmembrane domains of this family, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. The intracellular domain of this family, in contrast, exhibits significant diversity in length, amino acid composition, and function, and hence positions itself as a potential drug target. We therefore explored the interaction interface of the 5-HT3A intracellular domain (ICD) with its regulator, the resistance to inhibitors of choline esterase (RIC-3) protein. We have previously shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In this study, using synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay, we identified motif DWLRXX(X)VLDR, as a critical motif for interaction with RIC-3. This motif is repeated twice within the ICD. One site is within the MX-helix, another site is at the MAM4-helix transition. For both sites, triple-Ala substitutions (MX: W347, R349, and L353; MAM4: W447, R449, and L454) disrupt the interactions between 5-HT3A-ICD-peptide and RIC-3. Complementary studies using full-length 5-HT3A subunits confirmed that the Ala substitutions reduced the RIC-3 mediated modulation of 5-HT3A functional surface expression. We thus identified 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 Serotonin Receptor 3A (5-HT3A) is a member of the pentameric ligand-gated ion channel (pLGIC) superfamily and plays a role in numerous neuronal signaling pathways. However, relatively little remains known about its regulation. The intracellular domain (ICD) of the 5-HT3A receptor provides an attractive target for regulation studies, as the over 100 amino acid long sequence is exposed to the cell's internal milieu and is poorly conserved compared to that of other superfamily members. In this study, we set off to discover which proteins interact with the ICD of the 5-HT3A receptor, to identify potential regulatory partners. After developing a modified cellular fractionation technique using mouse brains, we were able to show, initially with western blotting, the presence of the 5-HT3A receptor in the endoplasmic reticulum (ER) where the receptor subunits assemble and the plasma membrane (PM) where the pentameric receptor is located. Once cellular localizations were preliminarily confirmed, proteins present in fractionated brain lysate fractions were identified using MALDI-TOF-MS/MS for further validation. Not only did this novel process work for serotonin receptors, it also worked for a variety of other key neurotransmitter receptors. These new techniques and findings provide a potential means of targeting regulatory proteins effecting numerous neuronal processes. This paves the way forward for progress in the study and treatment of neuropsychiatric disorders such as schizophrenia, Alzheimer's, and addiction.

Bupropion is an atypical antidepressant and smoking cessation drug which causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. However, the binding sites and binding mechanisms of bupropion are still elusive. To further understand the inhibition of pLGICs by bupropion, in this work, using a prokaryotic homologue of pLGICs as a model, we examined the inhibitory potency of bupropion in Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated ion channel. Bupropion inhibited proton-induced currents in GLIC with an inhibitory potency of 14.9 ± 2.0 μM, comparable to clinically attainable concentrations previously shown to also modulate eukaryotic pLGICs. Using single amino acid substitutions in GLIC and two-electrode voltage-clamp recordings, we further determined a binding site for bupropion in the lower third of the first transmembrane segment M1 at residue T214. The sidechain of M1 T214 together with additional residues of M1 and also of M3 of the adjacent subunit have previously been shown to contribute to binding of other lipophilic molecules like allopregnanolone and pregnanolone.