Expression and purification of a functional heteromeric GABAAreceptor for structural studies

Abstract

AbstractThe multi-subunit GABA-gated chloride channels of the Cys-loop receptor family, known as GABAAreceptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. In addition to their role in controlling synaptic tone, these receptors are the targets of a vast array of therapeutic compounds that potentiate channel gating. Importantly, functional activity and pharmacological efficacy of GABAAreceptors is coupled directly to the subunit composition. However, the absence of high resolution structural information precludes an explicit determination of the molecular mechanism of ligand binding to ion channel gating and modulation. Efforts to obtain this data are hindered largely by the lack of heterologous expression and purification protocols for high expressing receptor constructs. To address this issue, we describe a unique approach to identify bona fide functional GABAAreceptor subunit combinations by using theXenopusoocyte as an expression host in combination with fluorescence detection size exclusion chromatography. The results demonstrate that formation of a defined pentameric species is dependent on subunit composition. Furthermore, receptor subunits can tolerate large truncations in non-conserved M3/M4 cytoplasmic loop, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAAreceptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAAreceptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.