Aromatic Residues in the Transmembrane Helices Play an Essential Role in the Homopentameric Assembly of the Glucl α

Abstract

Pentameric ligand-gated ion channels (pLGICs) represent a major class of fast neurotransmitter receptors. In addition to nicotinic acetylcholine receptors (nAChRs), glycine receptors, GABAA receptors and 5HT3 receptors of vertebrates, pLGIC homologs have been identified in invertebrates and even in bacteria. Several crystal structures of pLGIC homologs have been solved at high resolution. By combining alanine scanning mutagenesis and homology modeling based on the X-ray structure of ELIC and the electron microscopy structure of the muscle-type nAChR, we could previously show that a network of aromatic residues within the transmembrane domains TM1, TM3 and TM4 is important for the proper assembly of human glycine receptor protomers into a functional homopentamer (Haeger et al. (2010), Nat Struct Mol Biol 17: 90-98). Here, we examined whether the TM helices of pLGIC homologs with solved crystal structure are also involved in homopentameric assembly. We chose the glutamate-gated chloride channel (GluCl) α, a pLGIC from the nematode Caenorhabditis elegans, which is most similar to the α1 glycine receptor and whose crystal structure was solved at 3.3 Å resolution (Hibbs and Gouaux (2011), Nature 474: 54-60). We performed an alanine scanning mutagenesis of all four TMs of GluCl α, purified the expressed GluCl mutants from Xenopus laevis oocytes by non-denaturing Ni2+-NTA chromatography, and analyzed them by blue native PAGE and SDS-PAGE. We found that aromatic residues in TM1, TM3 and TM4 are important for the correct homopentameric assembly of GluCl α protomers. These findings indicate that the role of aromatic residues in the assembly of chloride-conducting pLGICs is conserved across species.