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.

Serotonin type 3A receptors (5-HT 3A Rs) are cation-conducting homo-pentameric ligand-gated ion channels (pLGICs) also known as the Cys-loop superfamily in eukaryotes. 5-HT 3 Rs 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-HT 3A Rs, in structural features such as pentameric assembly.The intracellular domain of the mouse 5-HT 3A receptor (accession number: Q8K1F4) was generated as a fusion construct with N-terminal maltose binding protein (MBP) as we described previously (45) using the pMAL-c2x (New England Biolabs) variant pMALX (46). Based on this MBP-5-HT 3A -ICD template containing the entire wild-type ICD, deletions and substitutions were generated using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies), and confirmed by DNA sequencing (GENEWIZ, South Plainfield, NJ). The resulting amino acid sequences for all constructs are displayed in Figure 1. We will refer to the construct with the full-length ICD as ICD. The construct with the MA-helix deleted will be referred to as ∆ MA, the construct only consisting of the MA-helix will be MA, the construct with 44 amino acids between MX and MA removed will be ∆ 44, and finally the construct with three arginines mutated will be referred to as QDA. Expression and purification of the MBP-5-HT 3A -ICD constructsAll plasmids for expression of MBP-5-HT 3A -ICD fusion constructs with wild-type or engineered ICD were transformed into Escherichia coli (E. coli) BL21-CodonPlus-(DE3)-RIPL cells (Agilent Technologies). Cells were grown in Terrific Broth (TB) medium (1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, and 0.4% (v/v) glycerol) supplemented with ampicillin (100 µg/ml), chloramphenicol (34 µg/ml) and 0.2% (w/v) sterile glucose, at 37°C and 250 rpm, in a shaking incubator. All concentrations indicated are final concentrations unless otherwise stated. At OD 600 of 0.4-0.5, the cultures were induced by adding 0.4 mM isopropyl β -D-thiogalactoside (IPTG) and allowed to continue growth at 18°C for an additional 8 h. The cells were harvested by centrifugation at 4,600 g and 4 °C for 15 min, and then resuspended in (10 ml buffer/gram of the cell pellet) buffer A (20 mM Tris, pH 7.4, 200 mMNaCl, 1 mM TCEP, 2 mM EDTA) enriched with a freshly prepared...