ABSTRACT
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.
ABBREVIATIONS
- 5-HT3A
- serotonin type 3A
- BSA
- bovine serum albumin
- DNAse I
- deoxyribonuclease I
- E. coli
- Escherichia coli
- ECD
- extracellular domain
- EM
- electron microscopy
- GABAA
- γ-aminobutyric acid type A
- ICD
- intracellular domain
- IPTG
- Isopropyl β-D-1-thiogalactopyranoside
- MBP
- maltose-binding protein
- M3
- third transmembrane segment
- M4
- fourth transmembrane segment
- MA
- membrane-associate α-helix
- MBP-5-HT3A-ICD
- a chimera containing maltose-binding protein and the intracellular domain of serotonin type 3A receptors
- nAChRs
- nicotinic acetylcholine receptors
- PMSF
- phenylmethylsulfonyl fluoride
- pLGIC
- pentameric ligand-gated ion channel
- SEC
- size exclusion chromatography
- SEC-MALS
- size exclusion chromatography in line with multi-angle light scattering
- TCEP-HCl
- Tris(2-carboxyethyl)phosphine hydrochloride
- TMD
- transmembrane domain