Abstract
Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.
Highlights
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To screen for receptor function, we treated these transfected Human embryonic kidney (HEK) cells with increasing concentrations of a P2X7 agonist, Bz-ATP, and evaluated the capacity of this treatment to initiate a rapid low-divalent ative to the isotype control, HEK cells transfected with wild-type P2X7 exhibit a 4- to 10-fold increase in the geometric mean of the intensity of P2X7 cell surface immunostaining (Fig. 2A)
This increase in surface P2X7 immunostaining at room temperature is more substantial with HEK cells transfected with P2X7R578E/K579E (1.2 Ϯ 0.1-fold at 37°C vs 3.2 Ϯ 0.7-fold at room temperature; Fig. 2, B and D), suggesting that slowing down endocytic pathways can partially restore surface expression of this mutant
Summary
Microglia largely express the receptor as monomers, whereas peritoneal macrophages contain P2X7 multimers [17] This is likely to be specific to the cell type and/or differentiation state in that transfected fibroblasts do not appear to undergo P2X7-enhanced green fluorescence protein clustering in response to agonist administration [14]. In addition to participation in channel and pore activity, the P2X7 C terminus may direct receptor trafficking, consistent with observations showing that P2X4 C-terminal mutations can lead to constitutive surface localization [21] It is unclear whether P2X7 contains stabilization domains that interact with cell surface constituents or retention signals that prevent surface expression under certain conditions. The dibasic amino acid motif within the distal C-terminal portion of P2X7 contributes to receptor trafficking as well as ligand binding or channel gating
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