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Selective Knock-Down of P2X₇ ATP Receptor Function by Dominant-Negative Subunits

Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada (R.R., Y.C., D.B., A.S., P.S.); Unité Mixte de Recherche 5543, Centre National de la Recherche Scientifique, Université Victor Segalen Bordeaux 2, Bordeaux, France (E.B.-G.); and AstraZeneca R&D Charnwood, Loughborough, England (D.H.)

Among the family of P2X ATP-gated cation channels, the P2X₇ 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 P2X₇ produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X₇ 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 P2X₇ 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 P2X₇-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 P2X₇ receptors.

Address correspondence to: Dr. Philippe Séguéla, Montreal Neurological Institute, 3801 University Street, Suite 778, Montreal, QC, Canada, H3A 2B4. E-mail: philippe.seguela{at}mcgill.ca

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