Received for publication August 28, 2006.
Revised October 6, 2006.
Accepted for publication October 10, 2006.

Amino acid residues in the P2X₇ receptor that mediate differential sensitivity to ATP and BzATP

Abstract

Agonist properties of the P2X₇ receptor (P2X₇R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 µM) are required to activate the receptor and the ATP analogue, 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP), is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X₇R in order to delineate regions, or specific residues, that may be responsible for the unique actions of these agonists at the P2X₇R. We measured membrane currents in response to ATP and BzATP at wildtype rat and mouse P2X₇R, at chimeric P2X₇Rs, and at mouse P2X₇Rs bearing point mutations. Wildtype rP2X₇R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wildtype mP2X₇R. We found that agonist EC₅₀ values were determined solely by the ectodomain of the P2X₇R. Two segments (residues 115-136 and 282-288) when transposed together converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine²⁸⁴ in the rat P2X₇R, that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys¹²⁷ and Asn²⁸⁴ from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness, and also differentiate between the two widely used agonists at P2X₇ receptors.

Key words: Purinergic, Purinergic, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches

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