title: Role of the ρ1 GABA(C) receptor N-terminus in assembly, trafficking and function.
abstract: The GABAC receptor and closely related GABAA receptor are members of the pentameric ligand-gated ion channels (pLGICs) superfamily and mediate inhibitory fast synaptic transmission in the nervous system. Each pLGIC subunit comprises an N-terminal extracellular agonist-binding domain followed by a channel domain and a variable intracellular domain. Available structural information shows that the core of the agonist-binding domain is a β sandwich of ten β-strands, which form the agonist-binding pocket at the subunit interface. This β-sandwich is preceded by an N-terminal α-helix in eukaryotic structures but not in prokaryotic structures. The N-terminal α-helix has been shown to be functionally essential in α7 nicotinic acetylcholine receptors. Sequence analysis of GABAC and GABAA receptors predicts an α-helix in a similar position but preceded by 8 to 46 additional residues, of unknown function, which we term the N-terminal extension. To test the functional role of both the N-terminal extension and the putative N-terminal α-helix in the ρ1 GABAC receptor, we created a series of deletions from the N-terminus. The N-terminal extension was not functionally essential, but its removal did reduce both cell surface expression and cooperativity of agonist-gated channel function. Further deletion of the putative N-terminal α-helix abolished receptor function by preventing cell-surface expression. Our results further demonstrate the essential role of the N-terminal α-helix in the assembly and trafficking of eukaryotic pLGICs. They also provide evidence that the N-terminal extension, although not essential, contributes to receptor assembly, trafficking and conformational changes associated with ligand gating.
authors.affiliation: Health Innovation Research Institute, School of Medical Sciences, RMIT University , Bundoora, Victoria 3083, Australia.
authors.forename: Lik-Wei
authors.initials: LW
authors.lastname: Wong
authors.forename: Han-Shen
authors.initials: HS
authors.lastname: Tae
authors.forename: Brett A
authors.initials: BA
authors.lastname: Cromer
mesh_terms: D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D058446:Ligand-Gated Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: C087200:GABA-C receptor; D058446:Ligand-Gated Ion Channels; D018079:Receptors, GABA; D011978:Receptors, Nicotinic; D005680:gamma-Aminobutyric Acid
keywords: GABAC receptor; Ion channel; ligand-gating; microfluidic electrophysiology; protein trafficking