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 Saitoh, Tsuda Makoto, Kazuhide Inoue, Tadashi Ueda

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
JournalPurinergic Signalling
DOIs
Publication statusAccepted/In press - Jan 1 2019

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Purinergic P2X4 Receptors
Monoclonal Antibodies
Antibodies
Purinergic P2X Receptors
Head
Astrocytoma
Neuralgia
Site-Directed Mutagenesis
Ion Channels
Adenosine Triphosphate
Antigens
Amino Acids

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

Cite this

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure. / Igawa, Tatsuhiro; Kishikawa, Shuhei; Abe, Yoshito; Yamashita, Tomohiro; Nagai, Saki; Shiroishi, Mitsunori; Shinozaki, Chinatsu; Tanaka, Hiroyuki; Saitoh, Hidetoshi; Makoto, Tsuda; Inoue, Kazuhide; Ueda, Tadashi.

In: Purinergic Signalling, 01.01.2019.

Research output: Contribution to journalArticle

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AU - Abe, Yoshito

AU - Yamashita, Tomohiro

AU - Nagai, Saki

AU - Shiroishi, Mitsunori

AU - Shinozaki, Chinatsu

AU - Tanaka, Hiroyuki

AU - Saitoh, Hidetoshi

AU - Makoto, Tsuda

AU - Inoue, Kazuhide

AU - Ueda, Tadashi

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