Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos

• DOI: 10.1085/jgp.201812133
• Publication Date: 2019-04-15T19:05:47Z
• Authors: Vivian Wan Yu Liao; Han Chow Chua; Natalia Magdalena...; Mary Chebib; Thomas Balle; Philip Kiær Ahring
• Keywords: Concatemer; Cys-loop receptors; GABAA-rho receptor; Linker

γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter in mammalian central nervous system, are arguably most challenging member of pentameric Cys-loop to study due their heteromeric structure. When two or more subunits expressed together heterologous systems, variable subunit type, ratio, and orientation can form, precluding accurate interpretation data from functional studies. Subunit concatenation is a technique that involves linking individual theory allows precise control uniformity receptors. In reality, resulting concatemers widely used constructs flexible may therefore assemble with themselves free GABAAR unexpected ways. this study, we examine responses existing concatenated describe refinements necessary allow expression uniform receptor populations. We find dimers commonly constructs, β-23-α α-10-β, readily both clockwise counterclockwise orientations when coexpressed subunits. Furthermore, show formed new tetrameric α-10-β-α-β α-10-β-α-γ also give canonical αβγ To restrict linker flexibility, systematically shorten lengths dimeric optimized direct assembly GABAARs only one orientation, thus eliminating ambiguity associated previously described concatemers. Based on our data, revisit some noncanonical configurations proposed recent years explain how use have led wrong conclusions. Our results help clarify current contradictions literature regarding stoichiometry arrangement. The lessons learned guide future efforts understanding other related