Advantages of the Bungarotoxin Binding Site for Studying Live Membrane Protein Dynamics

A surprising approach to studying membrane receptors trafficking and mobility

Understanding the behavior of membrane proteins in live cells poses a significant challenge. Traditional methods often struggle to capture real-time processes or risk disrupting cellular functions. Antibodies and toxins, targeting the extracellular domain of membrane proteins, have been demonstrated to be useful research tools in many cases. However, a range of membrane proteins still lack such advanced tools, and demand alternative solutions. The bungarotoxin binding site (BBS) tagging technique, which hinges upon the high affinity of alpha-bungarotoxin (BTX), fills that gap and offers a solution that provides precision, as well as minimal invasiveness.

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The Science Behind BBS Tagging

To more easily study membrane protein dynamics, scientists have turned their attention to a toxin derived from the banded krait snake (Bungarus multicinctus), along with its binding site, normally found in the nicotinic acetylcholine receptor: BTX and the BBS. Rather than relying on large tags to track membrane proteins, this BBS tagging involves inserting the minimal BBS into proteins of interest. The short DNA sequence for BTX binding is incorporated into a construct which contains the sequence for the studied protein. It’s crucial that the BBS sequence be inserted within the sequence of the extracellular domain of the protein. You can then use fluorescently tagged BTX for specific labeling, allowing you to track protein dynamics. Originally developed for the nicotinic acetylcholine receptor (1), this method has since been adapted to look at a range of other receptors, including AMPA (2), GABAA (3) and GABAB (4) receptors, in live cells.

Advantages Over Traditional Methods

1. High specificity and affinity: BTX binds the BBS with nanomolar affinity, ensuring robust and specific labeling. This high affinity allows for accurate and reliable visualization of membrane proteins in live cells.
2. Minimal invasiveness: Traditional tags like GFP can be bulky (238 amino acids) with the potential to interfere with the protein’s natural behavior. In contrast, the BBS is only 13 amino acids long, making it significantly smaller and less likely to alter the protein’s function or mobility. This minimal size reduces the risk of adversely affecting cellular activity.
3. Versatility: BBS tagging has already been used with multiple membrane proteins, including AMPA receptor, calcium, potassium and Piezo1 channels, as well as GABAA and GABAB receptors (2–8). This versatility makes the BBS technique incredibly useful to anyone looking to study topics from synaptic transmission to receptor trafficking​​​​.
4. Compatibility with live cell imaging: A significant advantage of the BBS technique is that it allows researchers to observe and track the dynamics of membrane proteins in real-time – ideal for topics like receptor endocytosis and recycling in live neurons​​​​.
5. Reduced artifacts: Traditional methods like antibody labeling can introduce artifacts due to the size and multivalency of antibodies, which may result in cross-linking and clustering of target proteins. BBS tagging avoids these issues by using a small, monovalent tag that does not induce artificial aggregation of the labeled proteins. This leads to more accurate and reliable data on protein localization and dynamics​​.
6. Ease of use: The BBS technique is straightforward and efficient. Labeling with BTX is quick and does not require extensive optimization, unlike some other methods. Additionally, BTX is stable and easy to store, further simplifying the process

Alomone Labs’ Contributions

As membrane protein specialists, we have developed a range of products to be used with the BBS technique, including alpha-bungarotoxin conjugated to various fluorophores like ATTO Fluor 488 (#B-100-AG), ATTO Fluor 633 (#B-100-FR), FITC (#B-100-F), and biotin (#B-100-B). 

In a study published in the Journal of Visualized Experiments, the BBS technique was used to track GABAA receptor membrane localization and trafficking in hippocampal neurons (7). The study showcased the effectiveness of BBS tagging, demonstrating how fluorescently coupled BTX can be used to monitor successful trafficking of GABAA receptor​​. GABAA receptor are normally localized postsynaptically at inhibitory synapses; Figure 1 demonstrates localization of GABAA receptor with gephyrin – a postsynaptic marker for inhibitory synapses.

Figure 1. Confocal imaging of BBS and pHGFP tagged GABAAR showing appropriate localization with inhibitory synapse components. Surface GABAAR in α2pHGFP+BBS expressing neurons were labelled with α-bungarotoxin Alexa 594 (red), followed by immunostaining. Enlargements of dendrites are shown to the right. Merged panel (α-bungarotoxin Alexa 594 in red, GFP in green, gephyrin or in blue). Surface bungarotoxin labelled GABAAR show colocalization with the postsynaptic inhibitory scaffold protein gephyrin. (Scale bars, 10 μm.). Figure and legend from Brady et al. J Vis Exp, 2014 (7).

Alomone scientists put a lot of effort into validating our products and recommended protocols. Here, our scientists incorporated the BBS-tagged GABAB receptor 1 in HEK-293 cells, followed by labeling with fluorophore conjugated BTX (#B-100-FR) and anti-GABABR1 (#AGB-001-F). As shown below, BTX colocalized successfully with the GABAB receptor 1 (Figure 2).

Figure 2. Cells transfected with (A) GABAB receptor 1 or (B) bungarotoxin binding site (BBS)-tagged GABAB receptor 1 were fixed, followed by immunostaining with anti-GABABR1-FITC antibody (green, 1:5, #AGB-001-F), and labeled with α-bungarotoxin-ATTO Fluor-633 (red, 0.5 µM, #B-100-FR). 

Conclusion

The BBS tagging technique makes clever use of high-affinity binding between BTX and the BBS. It offers a minimally invasive, highly specific, and versatile method to study membrane protein dynamics in live cells. Using this BBS tagging approach also overcomes some of the main limitations of traditional methods, making it an interesting and effective experimental option. 

At Alomone Labs, you can find multiple α-bungarotoxin products along with an extensive range of membrane protein reagents. And if you ever need something more tailored, we have a Custom Service option for just that. 

References

1. M. J. Anderson, M. W. Cohen, Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle. J Physiol 237, 385-400.4 (1974). DOI: https://doi.org/10.1113/jphysiol.1974.sp010487.

2. Y. Sekine-Aizawa, R. L. Huganir, Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc Natl Acad Sci U S A 101, 17114–17119 (2004). DOI: https://doi.org/10.1073/pnas.0407563101

3.    Y. Bogdanov, G. Michels, C. Armstrong-Gold, P. G. Haydon, J. Lindstrom, M. Pangalos, S. J. Moss, Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO J 25, 4381–4389 (2006). DOI: https://doi.org/10.1038/sj.emboj.7601309.

4. M. E. Wilkins, X. Li, T. G. Smart, Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag. J Biol Chem 283, 34745–34752 (2008). DOI: https://doi.org/10.1074/jbc.M803197200.

5. G. T. Tabor, J. M. Park, J. G. Murphy, J.-H. Hu, D. A. Hoffman, A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking. Mol Cell Neurosci 98, 121–130 (2019). DOI: https://doi.org/10.1016/j.mcn.2019.06.007.

6. J. Wu, R. Goyal, J. Grandl, Localized force application reveals mechanically sensitive domains of Piezo1. Nat Commun 7, 12939 (2016). DOI: https://doi.10.1038/ncomms12939.

7.     M. L. Brady, C. E. Moon, T. C. Jacob, Using an α-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking. J Vis Exp, 51365 (2014). DOI: https://doi.org/10.3791/51365.

8. S. Hannan, M. E. Wilkins, P. Thomas, T. G. Smart, Tracking cell surface mobility of GPCRs using α-bungarotoxin-linked fluorophores. Methods Enzymol 521, 109–129 (2013). DOI: https://doi.org/10