Biology subjects

Stuebler, A. G.

Publications and source records attributed to Stuebler, A. G..

4 recordsLinked to original sources

bioRxiv2020

Mobility of lower MA-helices for ion-conduction through lateral portals in 5-HT3A 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 -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.

biophysics↗

bioRxiv2019

Bupropion inhibits serotonin type 3AB heteromeric channels at a physiologically relevant concentration

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 M and 505 M, respectively) but potentiated 5-HT-induced currents at low (30-50 M) concentrations. The corresponding IC50s for bupropion and hydroxybupropion with 5-HT3AR were 10- and 5-fold lower, respectively (87 M and 113 M), 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.\n\nSignificance 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.

pharmacology and toxicology↗

bioRxiv2019

Delineating the site of interaction on the intracellular domain of 5-HT3A receptors with the chaperone protein RIC-3

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.\n\nStatement 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.

biophysics↗

bioRxiv2019

Triple Arginines as Molecular Determinants for Pentameric Assembly of the Intracellular Domain of 5-HT3A Receptors

Serotonin type 3A receptors (5-HT3ARs) are cation-conducting homo-pentameric ligand-gated ion channels (pLGICs) also known as 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, schizophrenia, as well as chemotherapy-induced and post-operative 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 inside the ICD for pentameric assembly by first identifying the segments contributing to pentamerization using deletion constructs, and remarkably by making a small number of defined amino acid substitutions. Our work provides direct experimental evidence for the contribution of three arginines, previously implicated in governing the low conductance of 5-HT3ARs, in structural features such as pentameric assembly.

biophysics↗