Selective Knock-Down of P2X7 ATP Receptor Function by Dominant-Negative Subunits
2004
Among the family of P2X ATP-gated cation channels, the P2X 7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X 7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X 7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X 7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X 7 -dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2′,3′- O -(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 ± 9% and 25 ± 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X 7 receptors.
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