Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain
- LMU Munich, Germany
Abstract
The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing 'cap' domain, and a globular 'ballast domain' containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry (VCF). While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only three out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Original recordings or scans from SDS-PAGE gels are provided as source data and deposited with Dryad:This paper does not report original code.
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Table 1-source data 1Dryad Digital Repository, doi:10.5061/dryad.p8cz8w9tb.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (335447717 - SFB 1328,A15)
- Annette Nicke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Durner et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain
eLife 12:e82479.
https://doi.org/10.7554/eLife.82479
