Molecular characterization of purinergic receptor P2X4 involved in Japanese flounder (Paralichthys olivaceus) innate immune response and its interaction with ATP release channel Pannexin1

• ATP-gated purinergic P2X4 receptor gene (poP2X4R) was identified from Japanese Flounder P. olivaceus.
• poP2X4R mRNA distributes ubiquitously but with dominant expression in hepatopancreas tissue.
• poP2X4R is an immune response gene which is up-regulated by PAMPs and bacterial challenges.
• poP2X4R is a glycosylated membrane protein that interacts with Japanese flounder ATP release channel Pannexin1.

P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity.