Role of the ρ1 GABAC Receptor N-Terminus in Assembly, Trafficking and Function

The GABA(C) receptor and closely related GABA(A) receptor are members of the pentameric ligand-gated ion channels (pLGICs) superfarnily 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 beta sandwich of ten beta-strands, which form the agonist-binding pocket at the subunit interface. This beta-sandwich is preceded by an N-terminal alpha-helix in eukaryotic structures but not in prokaryotic structures. The N-terminal alpha-helix has been shown to be functionally essential in alpha 7 nicotinic acetylcholine receptors. Sequence analysis of GABA(C) and GABA(A) receptors predicts an alpha-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 alpha-helix in the rho l GABA(C) 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 alpha-helix abolished receptor function by preventing cell-surface expression. Our results further demonstrate the essential role of the N-terminal alpha-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.