Expression and purification of a functional heteromeric GABAA receptor for structural studies

PloS One
Derek P Claxton, Eric Gouaux

Abstract

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 ...Continue Reading

References

Aug 1, 1991·British Journal of Pharmacology·T G SmartR L Huganir
Apr 1, 1990·British Journal of Pharmacology·T G Smart, A Constanti
Oct 1, 1993·Current Opinion in Biotechnology·V A Luckow
Mar 1, 1996·Nature Biotechnology·A CrameriW P Stemmer
Apr 3, 1999·The Journal of Biological Chemistry·S J FarrarR M McKernan
Oct 16, 1999·Biochimica Et Biophysica Acta·W Weber
Sep 29, 2000·Biochemistry·H FukudaK Kuwajima
Jul 24, 2001·The Journal of Biological Chemistry·S W BaumannE Sigel
Sep 27, 2002·The Journal of Biological Chemistry·Sabine W BaumannErwin Sigel
Oct 9, 2002·Proceedings of the National Academy of Sciences of the United States of America·Philip J ReevesH Gobind Khorana
Dec 10, 2002·The Journal of Biological Chemistry·Karen BollanChristopher N Connolly
Jun 25, 2004·Nucleic Acids Research·Burkhard RostJinfeng Liu
Sep 24, 2004·Biotechnology and Bioengineering·Ching-Suei HsuYu-Chen Hu
Sep 29, 2004·Biochemical Pharmacology·Robert L MacdonaldJingqiong Kang
Feb 11, 2005·Journal of Molecular Biology·Nigel Unwin
Mar 2, 2005·Journal of Clinical Psychopharmacology·Kristin MitteMartin Hautzinger
Jun 17, 2005·Nature Reviews. Neuroscience·Delia Belelli, Jeremy J Lambert
Aug 1, 2006·Journal of Molecular Graphics & Modelling·Valérie Campagna-Slater, Donald F Weaver
Nov 7, 2006·Journal of Neurochemistry·Zi-Wei Chen, Richard W Olsen
Mar 7, 2008·Nature·Ricarda J C Hilf, Raimund Dutzler
Apr 4, 2008·Acta Neurologica Scandinavica·J RissS Collins
Apr 22, 2008·Nature Reviews. Neuroscience·Nicholas P Franks
Sep 2, 2008·Neuropharmacology·Richard W Olsen, Werner Sieghart
Sep 11, 2008·Protein Expression and Purification·Abhiram DukkipatiK Christopher Garcia
May 1, 2009·European Biophysics Journal : EBJ·Jennie M E CederholmTrevor M Lewis
Oct 27, 2009·BioTechniques·Ralph Hopkins, Dominic Esposito

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Methods Mentioned

BETA
X-ray
PCR
electrophoresis
size exclusion chromatography
flow cytometry
saturation binding
confocal microscopy
FRET
glycosylation
gel filtration

Software Mentioned

SignalP
pClamp
PredictProtein
ExPASy
GFPuv
Origin

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