Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization
- National Institute for Physiological Sciences, Japan
Abstract
Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, Figure 1- figure supplement 1, Figure 2, Figure 2 - figure supplement 1, Figure 3, Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, Figure 5 - figure supplement 1, Figure 5 - figure supplement 2, Figure 6, Figure 6 - figure supplement 1, and Figure 7
Article and author information
Author details
Rizki Tsari Andriani
Funding
Japan Society for the Promotion of Science (KAKENHI 17H04021)
- Yoshihiro Kubo
Japan Society for the Promotion of Science (KAKENHI 20H03424)
- Yoshihiro Kubo
Daiko Foundation
- Rizki Tsari Andriani
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were approved by the Animal Care Committee of the National Institutes of Natural Sciences (NINS, Japan) and performed obeying its guidelines.
Copyright
© 2021, Andriani & Kubo
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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Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization
eLife 10:e65822.
https://doi.org/10.7554/eLife.65822
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