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Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

Cys-loop receptors, also designated pentameric ligand-gated ion channels (pLGICs) include nicotinic acetylcholine receptors (nAChRs), serotonin type 3 receptors (5HT3Rs), γ-amino butyric acid type-A receptors (GABAARs) and glycine receptors (GlyRs). pLGICs function as obligate pentamers linked by non-covalent interactions between the N-terminal extracellular domains of identical or homologous subunits. Here we show that expression of GlyR α1 or 5HT3A subunits in two separate fragments (one containing the ectodomain and transmembrane domains M1-M3, and the other the fourth transmembrane domain M4) results in the assembly of functional pLGICs indistinguishable in their electrophysiological properties from wt pLGICs assembled from contiguous subunits. Alanine scanning of M1, M3 and M4 of the GlyR α1 subunit identified a total of 12 aromatic residues as important or crucial for pentameric assembly. The assembly-relevant aromatic residues cluster in one face of each helix. Homology modelling based on crystal structures (Hilf & Dutzler 2008; Bocquet et al 2008) predicted π- π interactions between the aromatic face of the M4 helix and three or two aromatic residues located in the M1 helix (Tyr228, Trp239, and Phe242) and the M3 helix (Trp286, Phe293), respectively. The loss of homopentamer formation and function seen upon alanine replacement of any of these contact residues strongly supports the existence of a membrane-embedded network of pairwisely interacting aromatic side chains that compacts and stabilizes the membrane core region of the GlyR. We infer from these results that a precise geometric arrangement of transmembrane helices defined by the tri-helical aromatic network is a prerequisite to allow the circular arrangement of the subunits stabilized essentially by earlier occurring random subunit interactions between the ectodomains.