No abstract

The cytolytic ionotropic ATP receptor P2X 7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X 7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X 4 , they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X 4 and P2X 7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X 4 and P2X 7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X 4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X 7 although there was no change in overall expression. Coexpression of a dominantnegative P2X 4 mutant (C353W) with P2X 7 , inhibited P2X 7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X 4 S341W with P2X 7 produced a current that was potentiated by ivermectin and inhibited by 2Ј, 3Ј-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X 7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X 4 and P2X 7 , which suggests that they associate to form heteromeric receptors.

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7 ؊/؊ mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ϳ4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.Acting via P2X receptors, ATP has several effects on immune cells, including triggering the release of pro-inflammatory cytokines, programmed cell death, and mycobacterial killing (1, 2). Until recently, attention has focused on the P2X7 receptor as the relevant sensor at the cell surface, and there is evidence for its involvement in neuropathic and inflammatory pain (3), arthritis (4), and the control of mycobacterial infection (5). P2X7 has a low affinity for ATP compared with the other predominant subtype expressed by immune cells, P2X4. The role of P2X4 receptors is less well understood, although in microglia they were shown to be up-regulated following peripheral nerve injury and to play an important role in the development of neuropathic pain (6). Regulation of the plasma membrane expression of these two receptors is not understood. Also, despite the considerable interest in both these receptor subtypes as potential targets in development of novel pain therapies, the subunit composition of the native receptors has not been determined.P2X receptor subunits associate to form trimeric complexes around a central conduction pore (7,8). With the exception of P2X6, they form functional homomeric receptors, and several subtypes also form functional heterotrimers (9). There is also evidence that P2X receptors form larger signaling complexes, interacting with other neigh...

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.