Expression, assembly and function of novel C‐terminal truncated variants of the mouse P2X7 receptor: re‐evaluation of P2X7 knockouts

Abstract

BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor‐mediated responses. Here, we investigated expression and function of C‐terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild‐type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)‐PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT‐PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in‐frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS‐PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist‐evoked whole cell currents were small. Co‐expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.

Loss of P2X7 nucleotide receptor function leads to abnormal fat distribution in mice

“…The current-voltage relationship of this conductance is consistent with that reported for P2X7 current in rabbit osteoclasts [38]. Although a splice variant (with C-terminal truncation) has been detected in some tissues of the Pfizer KO mouse, it is inefficiently trafficked to the cell surface and displays greatly diminished receptor function [39]. Thus, there appears to be global loss of P2X7 function in the Pfizer KO mouse model.…”

“…We used the Pfizer KO mouse in which there is global disruption of P2X7 function [25,39]. This mouse model has been studied extensively; however, most studies have focused on the phenotype of younger animals.…”

The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice

“…Collectively, this indicates P2X7 plays differing roles in various inflammatory skin disorders, but given previous findings [13,14], further investigation of P2X7 in human psoriasis and other mouse models of this disease is warranted. Moreover, it should be noted that C-terminal-truncated P2X7 variants are present at low amounts in C57BL/6 and Pfizer P2X7 KO mice [38], the same strains used in the current study. Although P2X7-induced pore formation is absent in splenic T and B cells [39], epidermal Langerhans cells and keratinocytes [20] from these P2X7 KO mice, a role for these escape P2X7 variants in IMQ-induced psoriasis-like inflammation in P2X7 KO mice cannot be excluded.…”

P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage

“…Though P2X7 is present in this mouse model, the protein is truncated at its C-terminus resulting in little-to-no receptor function [15]. Wild-type and knockout mouse colonies were maintained on a mixed genetic background (129/Ola × C57BL/6 × DBA/2).…”