Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.

Abstract

Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology 1-4 . This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes γ -aminobutyric acid receptors (GABA A Rs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators 3 . A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a β-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER) 5 in a vast range of potential subunit configurations 6 remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABA A R subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two α1 subunits of GABA A R each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of α1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for β2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric α7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.