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Published on: March 11, 2021
Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization
Yogesh Bhargava1, Annette Nicke, Jürgen Rettinger
1Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany. yogesh.bhargava@gmail.com
Abstract:
Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.
Insights
The P2X1 receptor
Area of Science:
- Neuroscience
- Molecular Biology
- Biophysics
Background:
- Ion channel function, including opening and desensitization, is crucial for neurotransmission.
- The ATP-gated P2X1 receptor (P2X1R) exhibits rapid and prolonged desensitization, complicating electrophysiological studies of its dynamics.
- Investigating agonist binding, unbinding, and recovery from desensitization in P2X1R is challenging.
Purpose of the Study:
- To develop a fluorescent tool for visualizing P2X1R dynamics.
- To elucidate the mechanisms underlying P2X1R desensitization and recovery.
- To investigate the role of agonist binding and unbinding in P2X1R desensitization.
Main Methods:
- Utilized Alexa-647-ATP as a fluorescent agonist for the P2X1R.
- Employed direct visualization techniques to monitor agonist binding and unbinding.
- Conducted experiments with competitive ligands to study unbinding kinetics.
- Developed computational models to analyze receptor desensitization.
Main Results:
- Alexa-647-ATP is a potent agonist for P2X1R, enabling direct visualization of its interactions.
- Long-lasting P2X1R desensitization results from slow agonist unbinding and agonist-induced receptor internalization.
- Competitive ligands accelerate Alexa-647-ATP unbinding from the desensitized receptor.
- Modeling suggests three agonist molecules are necessary for P2X1R desensitization, indicating a cooperative process.
Conclusions:
- Fluorescent agonists provide a powerful method for studying ion channel dynamics.
- P2X1R desensitization involves complex mechanisms including slow unbinding and internalization.
- Agonist unbinding from the desensitized P2X1R is a cooperative process influenced by ligand interactions.
