Biology subjects

Do, H. Q.

Publications and source records attributed to Do, H. Q..

3 recordsLinked to original sources

bioRxiv2023

Identification of a binding site for bupropion in Gloeobacter violaceus ligand-gated ion channel

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 M, 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. SIGNIFICANCEGLIC, 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, GABAA 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.

neuroscience↗

bioRxiv2022

Interaction Interface between 5-HT3A serotonin receptor and chaperone RIC-3

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 Alzheimers and Parkinsons 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.

neuroscience↗

bioRxiv2021

Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs

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. HighlightsO_LICell free expression of PCFT without any lipids or detergents resulted in quantitative precipitation of in-situ synthesized PCFT. C_LIO_LIAdditives for expression of PCFT in the soluble fraction were identified. C_LI

biochemistry↗