TY - JOUR
T1 - Expression and purification of a functional heteromeric GABAA receptor for structural studies
AU - Claxton, Derek P.
AU - Gouaux, Eric
N1 - Funding Information:
This work was supported by an NIH grant from the National Institute of General Medical Sciences (R01-GM100400) to E.G., who is also an Investigator with the Howard Hughes Medical Institute, and by an NRSA postdoctoral fellowship from the National Institute of General Medical Sciences (F32-GM100584) to D.P.C. The funders had no role in study design, data collection and The authors wish to thank Dr. David S Weiss of Baylor College of Medicine for providing the α1, β2 and γ2S genes in the pGEM vector. We thank Dr. David C Dawson for providing oocytes and Dr. Haining Zhong for the mKalama fluorescent protein. We also thank Dr. Stefanie Petrie of the OHSU Advanced Light Microscopy Core for expertise and data collection with the Zeiss LSM710 laser scanning confocal microscope. We thank Dr. Richard A Stein of Vanderbilt University for helpful discussions.
Publisher Copyright:
© 2018 Claxton, Gouaux. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2018/7
Y1 - 2018/7
N2 - The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor 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 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.
AB - The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor 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 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.
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U2 - 10.1371/journal.pone.0201210
DO - 10.1371/journal.pone.0201210
M3 - Article
C2 - 30028870
AN - SCOPUS:85051444522
VL - 13
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 7
M1 - e0201210
ER -