Triple Arginines as Molecular Determinants for Pentameric Assembly of the Intracellular Domain of 5-HT_3A Receptors

Abstract

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...

Mobility of Lower MA-Helices for Ion Conduction through Lateral Portals in 5-HT3A Receptors

“…A characteristic specific to the A subunit of 5-HT 3 Rs is that the MA-helices are lined with positively and negatively charged residues that interact to form inter-and intrasubunit salt bridges (37,38). Another notable salt bridge is located between the MA-helix and the L1-loop; D312 at the center of L1 is straddled by the 0 0 and 4 0 arginine residues (mouse R420 and R424, human R436 and R440) from the MA-helix of the adjacent subunit.…”

“…Another notable salt bridge is located between the MA-helix and the L1-loop; D312 at the center of L1 is straddled by the 0 0 and 4 0 arginine residues (mouse R420 and R424, human R436 and R440) from the MA-helix of the adjacent subunit. Amino acid substitutions introduced in the proximity of the five lateral portals, such as the QDA substitution, lead to the breakage of these salt bridges, which enhances flexibility of the MA-bundle, increases single-channel conductance, and disrupts pentamerization of the ICD (25,37,38). Based on these observations, we hypothesize that partial release of intersubunit salt bridges in the 5-HT 3A -QDA background yields a more open and unclamped or flexible arrangement of the ICD, in particular the MA-helices.…”

“…Previously, we have shown that the 5-HT 3A -ICD alone assembles into pentamers (18) and that disruption of salt bridges in conjunction with the QDA substitution also abolishes assembly of the 5-HT 3A -ICD into pentamers (38). In this study, we investigated the mobility of the lower end of the MA-helices (L402-R410, Fig.…”