Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits.

R. Raouf
Molecular Pharmacology. 2004-03-01; 65(3): 646-654
DOI: 10.1124/mol.65.3.646

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1. Mol Pharmacol. 2004 Mar;65(3):646-54.

Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits.

Raouf R(1), Chakfe Y, Blais D, Speelman A, Boué-Grabot E, Henderson D, Séguéla P.

Author information:
(1)Montreal Neurological Institute, Department of Neurology and Neurosurgery,
McGill University, Montreal, Quebec, Canada.

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.

DOI: 10.1124/mol.65.3.646
PMID: 14978243 [Indexed for MEDLINE]

Auteurs Bordeaux Neurocampus