Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

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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

The following previously published data sets were used

1. Mansoor et al
(2016) 5SVJ Crystal structure of the ATP-gated human P2X3 ion channel in the closed, apo state
Protein Data Bank, 5SVJ.

http://www.rcsb.org/3d-view/jsmol/5SVJ/1
1. Mansoor et al
(2016) 5SVK Crystal structure of the ATP-gated human P2X3 ion channel in the ATP-bound, open state
Protein Data Bank, 5SVK.

http://www.rcsb.org/3d-view/jsmol/5SVK/1
1. Brake et al
(1994) NM_053656 Amino acid sequence of rat P2X2
NCBI, NM_053656.3.

https://www.ncbi.nlm.nih.gov/nuccore/NM_053656

Article and author information

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Rizki Tsari Andriani

Biophysics and Neurobiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan

For correspondence
kiki@nips.ac.jp

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9242-469X
Yoshihiro Kubo

Biophysics and Neurobiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan

For correspondence
ykubo@nips.ac.jp

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-6707-0837

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.

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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.