Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization
- Division of Biophysics and Neurobiology, National Institute for Physiological Sciences, Japan
- Department of Physiological Sciences, The Graduate University for Advanced Studies, School of Life Science, Japan
Figures
Figure 1 with 3 supplements
Fluorescence signal changes of Anap-incorporated P2X2 receptor evoked by ATP and voltage.
(A) A scheme depicting the principle of the direct incorporation of fUAA (Anap) into the ion channel protein. The plasmid containing tRNA Anap-CUA and tRNA synthase is injected into the nucleus of Xe…
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Figure 1—source data 1
Fluorescence signal changes of A337Anap and I341Anap evoked by ATP and voltage.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
ATP-evoked currents of A337Anap and I341Anap were inhibited by P2X2 non-specific blockers: Suramin and PPADS.
(A–D) Representative current traces of P2X2 Anap constructs upon the application of 10 μM ATP (black) and 300 μM Suramin (dark blue); and 10 μM ATP (black) and 300 μM PPADS (purple), for A337Anap (A,…
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Figure 1—figure supplement 1—source data 1
ATP-evoked currents of A337Anap and I341Anap were inhibited by Suramin and PPADS.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Anap mutant scanning in the TM1 domain.
(A–S) Representative current and fluorescence recordings upon ATP and voltage application in Anap mutants located in the TM1 domain (VCF recordings were performed at least in three different batches …
Figure 1—figure supplement 3
Anap mutant scanning in the TM2 domain.
(A–T) Representative current and fluorescence recordings upon ATP and voltage application in Anap mutants located in the TM2 domain (VCF recordings were performed at least in three different batches …
Figure 2 with 1 supplement
Effect of SIK inhibitor treatment in Anap-incorporated Ci-VSP and P2X2 receptor.
SIK inhibitor treatment improved the VCF optical signal. (A–C) Representative fluorescence signal of VCF recordings of Ci-VSP without SIK inhibitor treatment, with 30 nM, and with 300 nM SIK …
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Figure 2—source data 1
Effect of SIK inhibitor treatment in Anap-incorporated Ci-VSP and P2X2 receptor.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig2-data1-v2.xlsx
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Figure 2—source data 2
Statistical analysis to support graph in Figure 2D.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig2-data2-v2.pdf
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Figure 2—source data 3
Statistical analysis to support graph in Figure 2E.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig2-data3-v2.pdf
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Figure 2—source data 4
Statistical analysis to support graph in Figure 2H.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig2-data4-v2.pdf
Figure 2—figure supplement 1
Effect of 300 nM SIK inhibitor application on the incidence of detectable Anap fluorescence signal change of P2X2 receptor.
(A, B) Incidence of detectable changes of Anap fluorescence for control group (57%, n = 7) and 300 nM SIK inhibitor application (80%, n = 12), respectively. Source data are provided in Figure …
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Figure 2—figure supplement 1—source data 1
Effect of 300 nM SIK inhibitor application on the incidence of detectable Anap fluorescence signal change of P2X2 receptor.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Voltage-clamp fluorometry (VCF) of Anap-incorporated P2X2 receptor in the presence of 300 nM SIK inhibitor upon ATP and voltage stimuli.
The focused electric field converged at A337 and I341 in TM2, throughout P2X2 ATP- and voltage-dependent gating. (A) Representative current traces and fluorescence signal of VCF recordings at A337, …
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Figure 3—source data 1
VCF of A337Anap and I341Anap in the presence of 300 nM SIK inhibitor upon ATP and voltage stimuli.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Voltage-clamp fluorometry (VCF) of L334Anap and L338Anap in the presence of 300 nM SIK inhibitor, with ATP and voltage steps.
(A, C) Representative current traces and fluorescence signal of VCF recordings at L334Anap and L338Anap, with 300 nM SIK inhibitor treatment, in the presence of 10 µM ATP (∆F/F = 0.38% ± 0.2 at 440 …
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Figure 3—figure supplement 1—source data 1
VCF of L334Anap and L338Anap in the presence of 300 nM SIK inhibitor, with ATP and voltage steps.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Voltage-clamp fluorometry (VCF) of Anap-labeled P2X2 at A337 in TM2 evoked by hyperpolarization in the absence and presence of ATP.
Anap fluorescence changes at A337 were observed even in the absence of ATP upon hyperpolarization. (A) Representative current traces and fluorescence signal of VCF recordings at A337 in the absence …
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Figure 4—source data 1
VCF of Anap-labeled P2X2 at A337 evoked by hyperpolarization in the absence and presence of ATP.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-data1-v2.xlsx
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Figure 4—source data 2
Statistical analysis to support graph in Figure 4B.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-data2-v2.pdf
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Figure 4—source data 3
Statistical analysis to support graph in Figure 4E.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-data3-v2.pdf
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Figure 4—source data 4
Statistical analysis to support graph in Figure 4H.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-data4-v2.pdf
Figure 4—figure supplement 1
VCF of Anap-labeled P2X2 at A337 in TM2 evoked by hyperpolarization in the absence and presence of ATP.
(A) Representative current traces of R313F upon application of 100 µM ATP. (B) Comparison of G-V relationships between R313F (turquoise filled triangle) and wildtype (black filled square) in 100 µM …
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Figure 4—figure supplement 1—source data 1
VCF of Anap-labeled P2X2 at A337 evoked by hyperpolarization in the absence and presence of ATP.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Statistical analysis to support graph in Figure 4—figure supplement 1G.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-figsupp1-data2-v2.pdf
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Figure 4—figure supplement 1—source data 3
Statistical analysis to support graph in Figure 4—figure supplement 1J.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig4-figsupp1-data3-v2.pdf
Figure 5 with 2 supplements
Voltage-clamp fluorometry (VCF) of Anap-labeled P2X2 at A337 in TM2 with the additional mutation of K308R evoked by hyperpolarization in the presence of ATP.
(A) Representative current traces and fluorescence signal of VCF recordings of K308R/A337Anap with 300 nM SIK inhibitor treatment in the presence of 300 µM ATP, from +40 mV to −160 mV with a holding …
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Figure 5—source data 1
VCF of K308R/A337Anap evoked by hyperpolarization in the presence of ATP.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
ATP-evoked currents in K308R/A337Anap were inhibited by P2X2 non-specific blockers: Suramin and PPADS.
(A–B) Representative current traces of P2X2 Anap constructs upon the application of 10 μM ATP (black) and 300 μM Suramin (dark blue); and 10 μM ATP (black) and 300 μM PPADS (purple), respectively. …
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Figure 5—figure supplement 1—source data 1
ATP-evoked currents of K308R/A337Anap were inhibited by Suramin and PPADS.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Slow fraction of fluorescence changes at A337 in TM2 with the additional mutation of K308R, evoked by hyperpolarization, was [ATP]-dependent.
(A) Representative current traces and fluorescence signal of VCF recordings of K308R/A337Anap with 300 nM SIK inhibitor treatment in the presence of 30 µM ATP, from +40 mV to −160 mV with a holding …
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Figure 5—figure supplement 2—source data 1
Slow fraction of fluorescence changes at K308R/A337Anap, evoked by hyperpolarization, was [ATP] dependent.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig5-figsupp2-data1-v2.xlsx
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Figure 5—figure supplement 2—source data 2
Statistical analysis to support the graph in Figure 5—figure supplement 2G.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig5-figsupp2-data2-v2.pdf
Figure 6 with 1 supplement
Voltage-clamp fluorometry (VCF) of Anap-labeled P2X2 at A337 in TM2 with the additional mutation of K308R, evoked by hyperpolarization in the absence and presence of ATP.
Fluorescence signal changes at K308R/A337Anap exhibited only a fast component in the absence of ATP and consisted of two components in the presence of ATP. (A) Representative current traces and …
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Figure 6—source data 1
VCF of K308R/A337Anap evoked by hyperpolarization in the absence and presence of ATP.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Voltage-clamp fluorometry (VCF) of Anap-labeled P2X2 at A337 in TM2 with the additional mutation of K308R, evoked by hyperpolarization in the absence and presence of ATP.
(A) Representative current traces and fluorescence signal of VCF recordings of K308R/A337Anap in the absence of ATP (∆F/F = 4.2% ± 1.01 at 440 nm; n = 5) and 300 µM ATP (∆F/F = 2.1% ± 0.5; n = 5). (B…
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Figure 6—figure supplement 1—source data 1
VCF of K308R/A337Anap evoked by hyperpolarization in the absence and presence of ATP.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig6-figsupp1-data1-v2.xlsx
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Figure 6—figure supplement 1—source data 2
Statistical analysis to support graph in Figure 6—figure supplement 1B.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig6-figsupp1-data2-v2.pdf
Figure 7 with 1 supplement
Effects of mutations at A337 in TM2 and F44 in TM1 on P2X2 receptor ATP- and voltage-dependent gating.
(A) Representative current traces of single amino acid mutants at the position of A337 in the presence of 30 µM ATP, in response to voltage step pulses from +40 mV to −140 mV, with a holding …
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Figure 7—source data 1
Effects of mutations at A337 in TM2 and F44 in TM1 on P2X2 receptor ATP- and voltage-dependent gating.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig7-data1-v2.xlsx
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Figure 7—source data 2
Statistical analysis to support graph in Figure 7I.
- https://cdn.elifesciences.org/articles/65822/elife-65822-fig7-data2-v2.pdf
Figure 7—figure supplement 1
Effect of swapped mutation F44A/A337F.
(A–F) Representative current traces of F44A/A337F upon various [ATP] application (1, 3, 10, 30, 100, 300 µM), followed by voltage application at each concentration (n = 3). Voltage-dependent gating …
Figure 8
Proposed initiation mechanisms of P2X2 receptor complex gating.
(A, B) Top view structure of the P2X2 receptor in the closed (A) and ATP-bound open state (B). Depicted are the proposed initiation mechanisms of P2X2 receptor complex gating as follows. (1) The …
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | Rattus norvegicus P2X2 | Brake et al., 1994 | ||
| Gene (Ciona intestinalis) | Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) | Sakata et al., 2016 | ||
| Strain, strain background (Escherichia coli) | XL1-Blue | Agilent Technologies | ||
| Strain, strain background (Escherichia coli) | TG1 | Clontech | ||
| Recombinant DNA reagent | pAnap (plasmid) | Addgene | Plasmid #48696 | cDNA encoding the tRNA synthetase/Anap-CUA |
| Commercial assay or kit | QuikChange II site-directed mutagenesis | Agilent Technologies | 200524 | |
| Commercial assay or kit | mMESSAGE T7 RNA transcription kit | Thermo Fisher Scientific | AM1344 | |
| Commercial assay or kit | mMESSAGE SP6 RNA transcription kit | Thermo Fisher Scientific | AM1340 | |
| Chemical compound, drug | 0.15% tricaine | Sigma-Aldrich | ||
| Chemical compound, drug | Collagenase type 1 | Sigma-Aldrich | ||
| Chemical compound, drug | ATP disodium salt | Sigma-Aldrich | 34369-07-8 | |
| Chemical compound, drug | Anap sodium salt | FutureChem Chemicals | FC-8101 | |
| Chemical compound, drug | HG 9-91−01/SIK inhibitor | MedChem Express | 1456858-58-4 | |
| Chemical compound, drug | Suramin sodium salt | Sigma-Aldrich | 129-46-4 | |
| Chemical compound, drug | PPADS tetrasodium salt | Sigma-Aldrich | P178 | |
| Software, algorithm | Igor Pro 5.01 | Wavemetrics | RRID:SCR_000325 | |
| Software, algorithm | PyMOL Molecular Graphics System ver. 2.3.0 | Schrodinger LLC | RRID:SCR_000305 | |
| Software, algorithm | OriginPro | OriginLab | RRID:SCR_014212 | |
| Software, algorithm | GraphPad Prism 9 | GraphPad Software, Inc. | RRID:SCR_002798 | |
| Software, algorithm | SWISS-MODEL | Arnold et al., 2006; Biasini et al., 2014 | RRID:SCR_018123 | |
| Software, algorithm | Protter protein visualization | Omasits et al., 2014 | https://wlab.ethz.ch/protter/start/ | |
| Software, algorithm | BioRender | BioRender.com | RRID:SCR_018361 | Figure 1A created with BioRender |
Additional files
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Supplementary file 1
List of introduced TAG mutations in P2X2 receptor for VCF analysis.
Mutations were introduced one at a time into 96 positions within the extracellular domain (ECD) near the ATP-binding site and extracellular linker, transmembrane domains (TMs), intracellular N-terminal, and intracellular C-terminal. ATP application ranging from 10 µM, 30 µM, or 100 µM unless otherwise stated. (+) indicates there was either ATP-evoked fluorescence (F) signal change, voltage-evoked F change, ATP-evoked current (I) change, or voltage-evoked I change. (-) indicates negative results. (**) indicates mutants which have a very low expression level, so that the reliable VCF analysis could not be undertaken. (***) indicates fast current decay. (--) indicates that the subsequent recording could not be performed, as a result of fast current decay. (n.d.) indicates not determined.
- https://cdn.elifesciences.org/articles/65822/elife-65822-supp1-v2.docx
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Transparent reporting form
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