title: Using an α-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking.
abstract: It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.
authors.affiliation: Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Brady
authors.affiliation: Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Moon
authors.affiliation: Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine; tcj11@pitt.edu.
authors.forename: Tija C
authors.initials: TC
authors.lastname: Jacob
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000737:chemistry* / Q000378:metabolism; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*
chemical_list: C417664:Alexa594; D002038:Bungarotoxins; D009930:Organic Chemicals; D011963:Receptors, GABA-A
references.citation: Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594-8
references.pmid: 1070010
references.citation: Mol Pharmacol. 2009 Nov;76(5):992-7
references.pmid: 19666700
references.citation: J Neurosci. 2005 Nov 9;25(45):10469-78
references.pmid: 16280585
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12500-5
references.pmid: 19617557
references.citation: J Biol Chem. 2008 Dec 12;283(50):34745-52
references.pmid: 18812318
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 3;107(31):13918-23
references.pmid: 20643948
references.citation: EMBO J. 2012 Jun 29;31(13):2937-51
references.pmid: 22531784
references.citation: J Physiol. 1974 Mar;237(2):385-400
references.pmid: 4133039
references.citation: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6629-34
references.pmid: 11381118
references.citation: J Biol Chem. 2011 Jul 8;286(27):24324-35
references.pmid: 21724853
references.citation: J Biol Chem. 2008 Jul 4;283(27):18538-44
references.pmid: 18467327
references.citation: Biophys Chem. 2003;100(1-3):293-305
references.pmid: 12646372
references.citation: Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):18595-600
references.pmid: 23091016
references.citation: Nat Neurosci. 2005 Jul;8(7):889-97
references.pmid: 15951809
references.citation: EMBO J. 2006 Sep 20;25(18):4381-9
references.pmid: 16946701
references.citation: Neuron. 2009 Jun 11;62(5):670-82
references.pmid: 19524526
references.citation: Nature. 1998 Jul 9;394(6689):192-5
references.pmid: 9671304
references.citation: Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16679-84
references.pmid: 20823221
references.citation: Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17114-9
references.pmid: 15563595
references.citation: Proc Natl Acad Sci U S A. 1980 Aug;77(8):4823-7
references.pmid: 6933533