Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure

  • Tatsuhiro Igawa
  • Shuhei Kishikawa
  • Yoshito Abe
  • Tomohiro Yamashita
  • Saki Nagai
  • Mitsunori Shiroishi
  • Chinatsu Shinozaki
  • Hiroyuki Tanaka
  • Hidetoshi Tozaki-Saitoh
  • Makoto Tsuda
  • Kazuhide InoueEmail author
  • Tadashi UedaEmail author


P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4’s head domain (rHD, Gln111–Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.


P2X4 receptor Neuropathic pain Monoclonal antibody FSEC 



Circular dichroism


Complete Freund’s adjuvant


Extracellular domain of P2X4 receptor


Incomplete Freund’s adjuvant


Keyhole limpet hemocyanin


Phosphate-buffered saline


Rat head domain of P2X4 receptor


Trimethylammonium propyl sulfonate



We thank Dr. Jose Caaveiro of Kyshu University for critical readership of the manuscript. ITC measurements were supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Funding information

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to UT (23390155) and YA (18K06597) and by a grant from the Japan Foundation for Applied Enzymology.

Compliance with ethical standards

Conflicts of interest

Tatsuhiro Igawa declares that he has no conflict of interest.

Shuhei Kishikawa declares that he has no conflict of interest.

Yoshito Abe declares that he has no conflict of interest.

Tomohiro Yamashita declares that he has no conflict of interest.

Saki Nagai declares that she has no conflict of interest.

Mitsunori Shiroishi declares that he has no conflict of interest.

Chinatsu Shinozaki declares that he has no conflict of interest.

Hiroyuki Tanaka declares that he has no conflict of interest.

Hidetoshi Tozaki-Saitoh declares that he has no conflict of interest.

Makoto Tsuda declares that he has no conflict of interest.

Kazuhide Inoue declares that he has no conflict of interest.

Tadashi Ueda declares that he has no conflict of interest.

Ethical approval

All animal experiments were conducted according to the relevant national and international guidelines in the Act on Welfare and Management of Animals (Ministry of Environment of Japan) and the Regulation of Laboratory Animals (Kyushu University), and under the protocols approved by the Institutional Animal Care and Use Committee review panels at Kyushu University.

Supplementary material

11302_2019_9646_MOESM1_ESM.docx (107 kb)
ESM 1 (DOCX 107 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Tatsuhiro Igawa
    • 1
  • Shuhei Kishikawa
    • 1
  • Yoshito Abe
    • 1
  • Tomohiro Yamashita
    • 2
  • Saki Nagai
    • 1
  • Mitsunori Shiroishi
    • 1
  • Chinatsu Shinozaki
    • 1
  • Hiroyuki Tanaka
    • 3
  • Hidetoshi Tozaki-Saitoh
    • 4
  • Makoto Tsuda
    • 4
  • Kazuhide Inoue
    • 5
    Email author
  • Tadashi Ueda
    • 1
    Email author
  1. 1.Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan
  2. 2.Department of Global Health-Care, Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan
  3. 3.Department of Pharmacognosy Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan
  4. 4.Department of Life Innovation, Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan
  5. 5.Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan

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