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Are the putative N-terminal α-helix and preceding residues important in GABAA/C receptor function?

L.W. Wong,1,2 H.-S. Tae1 and B.A. Cromer,1 1Health Innovation Research Institute, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia and 2Department of Pharmacology, University of Melbourne, Parkville, VIC 3010, Australia.

The GABAA/C receptors are members of the pentameric Cys-loop superfamily of ligand-gated ion channels (pLGICs) and mediate inhibitory fast synaptic transmission in the nervous system. pLGICs comprise an N-terminal extracellular agonist-binding domain followed by a channel domain and intracellular domain. Available structural information shows the agonist binding domain comprises a β sandwich of ten β-strands, which form the agonist binding pocket, preceded by an N-terminal α-helix in eukaryotic structures, not present in prokaryotic structures. Sequence analysis of GABAA/C receptors predicts an α-helix in a similar position. This putative α-helix in GABAA/C receptors is, however, preceded by 10 to 46 additional residues not present in other pLGICs, which we term the N-terminal extension. The N-terminal α-helix has been shown to be functional essential in nicotinic acetylcholine receptors (Bar-Lev et al., 2011; Castillo et al., 2009). The role of the α-helix and N-terminal extension in GABAA/C receptors has not been tested and is the subject of this study. We found in both homomeric ρ1 GABAC and heteromeric α1β2γ2 GABAA receptors that the putative α-helix is essential for receptor function but not the N-terminal extension. Partial or complete removal of the N-terminal extension in ρ1 GABAC receptors did, however, affect cell surface expression and agonist sensitivity. Mutation of a putative furin protease cleavage motif within the N-terminal extension of ρ1 GABAC receptors likewise impaired cell surface expression. Our results support the essential role of the N-terminal α-helix in eukaryotic pLGICs and provide evidence that the N-terminal extension has a subtle modulatory role on receptor function.

Castillo M, Mulet J, Aldea M, Gerber S, Sala S, Sala F, Criado M. (2009) Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors. Journal of Neurochemistry 108: 1399-1409.

Bar-Lev DD, Degani-Katzav N, Perelman A, Paas Y. (2011) Molecular dissection of a Cl-selective Cys-loop receptor points to components that are dispensable or essential for channel activity. Journal of Biological Chemistry 286: 43830-41.