Support was provided by startup-funds from the Pharmacology and Chemical Biology Department at the University of Pittsburgh School of Medicine. Acknowledgement of Jacob lab members who contributed to the video submission: Nicholas Graff.

All protocols described below are in accordance with the IACUC and IRB institutional review boards of the University of Pittsburgh School of Medicine.
1. Preparation of Hippocampal Neuronal Cultures in Tissue Culture Hood
Note: Use sterile technique and reagents throughout Protocol&#160;1.
<ol>
	<li>Prepare poly-D-lysine (0.1 mg/ml in H2O) coated glass coverslips as a substrate for the neuronal culture.
	<ol>
		<li>Place 4-5 round glass coverslips inside each 3.5&#160;cm ti...

<ol>
	<li>Jacob, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gephyrin+regulates+the+cell+surface+dynamics+of+synaptic+GABAA+receptors.">Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.</a> J. Neurosci. 25, 10469-10478 (2005).</li><li>Bogdanov, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+GABAA+receptors+are+directly+recruited+from+their+extrasynaptic+counterparts.">Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts.</a> EMBO J. 25, 4381-4389 (2006).</li><li>Thomas, P., Mortensen, M., Hosie, A. M., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+mobility+of+functional+GABA(A)+receptors+at+inhibitory+synapses.">Dynamic mobility of functional GABA(A) receptors at inhibitory synapses.</a> Nat. Neurosci. 8, 889-897 (2005).</li><li>Wilkins, M. E., Li, X., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Cell+Surface+GABAB+Receptors+Using+an+&#945;-Bungarotoxin+Tag.">Tracking Cell Surface GABAB Receptors Using an &#945;-Bungarotoxin Tag.</a> J. Biol. Chem. 283, 34745-34752 (2008).</li><li>Saliba, R. S., Pangalos, M., Moss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-like+protein+Plic-1+enhances+the+membrane+insertion+of+GABAA+receptors+by+increasing+their+stability+within+the+endoplasmic+reticulum.">The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum.</a> J. Biol. Chem. 283, 18538-18544 (2008).</li><li>Bannai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-Dependent+Tuning+of+Inhibitory+Neurotransmission+Based+on+GABAAR+Diffusion+Dynamics.">Activity-Dependent Tuning of Inhibitory Neurotransmission Based on GABAAR Diffusion Dynamics.</a> Neuron. 62, 670-682 (2009).</li><li>Muir, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+regulate+GABAA+receptor+lateral+mobility+and+clustering+at+inhibitory+synapses+through+serine+327+on+the+gamma2+subunit.">NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the gamma2 subunit.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 16679-16684 (2010).</li><li>Katchalski-Katzir, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+peptides+that+bind+alpha-bungarotoxin+with+high+affinity+and+mimic+the+three-dimensional+structure+of+the+binding-site+of+acetylcholine+receptor.">Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor.</a> Biophys. Chem. 100, 293-305 (2003).</li><li>Scherf, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beta+-hairpin+structure+in+a+13-mer+peptide+that+binds+alpha+-bungarotoxin+with+high+affinity+and+neutralizes+its+toxicity.">A beta -hairpin structure in a 13-mer peptide that binds alpha -bungarotoxin with high affinity and neutralizes its toxicity.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 6629-6634 (2001).</li><li>Wilkins, M. E., Li, X., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+cell+surface+GABAB+receptors+using+an+alpha-bungarotoxin+tag.">Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag.</a> J. Biol. Chem. 283, 34745-34752 (2008).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Jacob, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzodiazepine+treatment+induces+subtype-specific+changes+in+GABAA+receptor+trafficking+and+decreases+synaptic+inhibition.">Benzodiazepine treatment induces subtype-specific changes in GABAA receptor trafficking and decreases synaptic inhibition.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 18595-18600 (2012).</li><li>Anderson, M. J., Cohen, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+staining+of+acetylcholine+receptors+in+vertebrate+skeletal+muscle.">Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle.</a> J. Physiol. 237, 385-400 (1974).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinkage+and+visualization+of+acetylcholine+receptors+on+myotubes+with+biotinylated+alpha-bungarotoxin+and+fluorescent+avidin.">Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.</a> Proc. Natl. Acad. Sci. U.S.A. 77, 4823-4827 (1980).</li><li>Axelrod, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+motion+of+fluorescently+labeled+acetylcholine+receptors+in+membranes+of+developing+muscle+fibers.">Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.</a> Proc. Natl. Acad. Sci. U.S.A. 73, 4594-4598 (1976).</li><li>Sekine-Aizawa, Y., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+receptor+trafficking+by+using+alpha-bungarotoxin-binding-site-tagged+receptors.">Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17114-17119 (2004).</li><li>Jacob, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA+receptor+membrane+trafficking+regulates+spine+maturity.">GABAA receptor membrane trafficking regulates spine maturity.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 12500-12505 (2009).</li><li>Hannan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAB+receptor+internalisation+is+regulated+by+the+R2+subunit.">GABAB receptor internalisation is regulated by the R2 subunit.</a> J. Biol. Chem. , (2011).</li><li>Saliba, R. S., Kretschmannova, K., Moss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+phosphorylation+of+GABAA+receptors+regulates+receptor+insertion+and+tonic+current.">Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current.</a> EMBO. 31, 2937-2951 (2012).</li><li>Beqollari, D., Betzenhauser, M. J., Kammermeier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+G-Protein+Coupling+in+an+mGluR6+Point+Mutant+Associated+with+Congenital+Stationary+Night+Blindness.">Altered G-Protein Coupling in an mGluR6 Point Mutant Associated with Congenital Stationary Night Blindness.</a> Mol. Pharmacol. 76, 992-997 (2009).</li><li>Terunuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+activation+of+NMDA+receptors+promotes+dephosphorylation+and+alters+postendocytic+sorting+of+GABAB+receptors.">Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 13918-13923 (2010).</li><li>Goslin, K. G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Nerve+Cells.">Culturing Nerve Cells.</a> , (1998).</li></ol>

The authors have nothing to disclose.

The BBS based fixed and live techniques described here can be used to track receptor or other plasma membrane protein trafficking in cell lines, neurons, and other primary cells. This method has been successfully used to study membrane insertion and removal of ligand-gated ion channels and GPCR and assess changes in trafficking due to the presence of receptor agonists and modulators. Key aspects include appropriate localization of the tag to an extracellular location and performing controls to ensure that addition of the...

The use of fluorescently coupled &#945;-bungarotoxin to study receptor localization and dynamics was pioneered in studies of the nicotinic acetylcholine receptor13-15, the toxin&#39;s endogenous target. Subsequently, incorporation of the minimal Bgt binding peptide (BBS) has been used to study trafficking of both excitatory and inhibitory ligand-gated ion channels and G protein coupled receptors2,10,16-21. This BBS-based technique provides advantages to other approaches used for trafficking studies such as surface biotinylation methods, antibody labeling of live cells with antibodies to extracellular epitopes, and fluorescence recovery after photobleaching (FRAP). During cell surface biotinylation free amines are covalently modified, with the potential to affect cellular activity. Antibody based studies have often been hampered by surface antigen clustering or capping, which can alter trafficking events. Due to the bleaching step for FRAP studies, an important concern is damaging the underlying cellular structure. An additional advantage is that BBS tagged constructs can also be used for biochemical methodologies with biotin-coupled bungarotoxin to asses receptor trafficking. This technique is readily applicable to cell lines and primary cells. For use in cells that express nicotinic acetylcholine (nAChR) receptors, the nAChR antagonist tubocurarine must be used throughout the protocol as indicated. Performing a simple surface Bgt labeling (equivalent to the T=0 time point for the endocytosis protocol) on untransfected cells in the absence of tubocurarine will provide evidence of endogenous nAChR.
An important consideration for using this technique is appropriate insertion of the BBS so that it is present at an extracellular location when the protein of interest is delivered to the plasma membrane. For example, the N-terminal domains of GABAAR subunits reside in the vesicular lumen during trafficking and become extracellular after receptor insertion in the plasma membrane, allowing specific labeling of cell surface receptors and assessment of their removal from the cell surface by endocytic events. We have previously shown that the addition of GFP, myc, or BBS epitopes to this domain of GABAAR subunits is functionally silent. Standard controls should be performed to ensure that the tagged protein is expressed at similar levels to an untagged construct, that it appropriately localized, and that it does not affect receptor function. This characterization of transfected constructs&#160;will also aid in troubleshooting overexpression concerns.

<table border="0" cellpadding="0" cellspacing="0" style="width:811px;" width="809">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
		</tr>
	</tbody>
</table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. 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 receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) 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&#39; 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.

Characterization of a BBS tagged construct includes important controls such as determining if the expressed protein assembles properly (particularly with receptors composed of multiple subunits), traffics to the cell surface and localizes appropriately. Inhibitory synapses are composed of GABA A receptor surface clusters that colocalize with the inhibitory scaffold protein gephyrin and are apposed to presynaptic inhibitory terminals, identified by the vesicular inhibitory amino acid transporter that loads GABA and glycin...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Research

JoVE Journal

Neuroscience

15.7K Views.  University of Pittsburgh School of Medicine. Here we demonstrate the use of fluorescent Alexa dye coupled to α-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.