title: Assembly, trafficking and function of α1β2γ2 GABAA receptors are regulated by N-terminal regions, in a subunit-specific manner.
abstract: GABAA receptors are pentameric ligand-gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand-gated ion channels structures, sequence analysis predicts an α-helix near the N-terminus of each GABAA receptor subunit. Preceding each α-helix are 8-36 additional residues, which we term the N-terminal extension. In homomeric GABAC receptors and nicotinic acetylcholine receptors, the N-terminal α-helix is functionally essential. Here, we determined the role of the N-terminal extension and putative α-helix in heteromeric α1β2γ2 GABAA receptors. This role was most prominent in the α1 subunit, with deletion of the N-terminal extension or further deletion of the putative α-helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N-terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α-helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α-helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N-terminal extensions and α-helices make key subunit-specific contributions to assembly, consistent with both regions being involved in inter-subunit interactions. N-terminal α-helices and preceding sequences of eukaryotic pentameric ligand-gated ion channels are absent in prokaryotic homologues, suggesting they may not be functionally essential. Here, we show that in heteropentameric α1β2γ2 GABAA receptors, the role of these segments is highly subunit dependent. The extension preceding the α-helix in the α subunit is crucial for assembly and trafficking, but is of little importance in β and γ subunits. Indeed, robust receptor levels remain when the extension and α-helix are removed in β or γ subunits.
authors.affiliation: Health Innovation Research Institute, School of Medical Sciences, RMIT University, Melbourne, Vic., Australia.|Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, Vic., Australia.
authors.forename: Lik-Wei
authors.initials: LW
authors.lastname: Wong
authors.affiliation: Health Innovation Research Institute, School of Medical Sciences, RMIT University, Melbourne, Vic., Australia.
authors.forename: Han-Shen
authors.initials: HS
authors.lastname: Tae
authors.affiliation: Health Innovation Research Institute, School of Medical Sciences, RMIT University, Melbourne, Vic., Australia.
authors.forename: Brett A
authors.identifier: http://orcid.org/0000-0003-0743-1535
authors.initials: BA
authors.lastname: Cromer
mesh_terms: D000595:Amino Acid Sequence; D016384:Consensus Sequence; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D011963:Receptors, GABA-A / Q000737:chemistry / Q000172:deficiency / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D009435:Synaptic Transmission / Q000502:physiology; D015032:Zinc / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: C546240:GABRA1 protein, human; C495710:GABRB2 protein, human; C546279:GABRG2 protein, human; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid; D015032:Zinc
keywords: GABAA receptor; ligand-gated ion channels; receptor assembly; trafficking