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 Inoue, Tadashi Ueda

Research output: Contribution to journalArticle

2 Citations (Scopus)

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
Pages (from-to)27-35
Number of pages9
JournalPurinergic Signalling
Volume15
Issue number1
DOIs
Publication statusPublished - Mar 15 2019

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

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