A Functional P2X7 Splice Variant with an Alternative Transmembrane Domain 1 Escapes Gene Inactivation in P2X7 Knock-out Mice

Annette Nicke,Yung-Hui Kuan,Marianela Masin,Jürgen Rettinger,Benjamin Marquez-Klaka,Olaf Bender,Dariusz C Górecki,Ruth D Murrell-Lagnado,Florentina Soto

doi:10.1074/jbc.m109.033134

Abstract

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-D-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7(-/-) mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7(-/-) strains has important implications for our understanding of the role of this receptor in health and disease.

Highlights

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target
The P2X7 receptor is distinguished from other P2X receptors by its long intracellular C terminus, a low ATP sensitivity (EC50: 100 ␮M to 1 mM), and its ability to induce “cell permeabilization,” meaning that upon prolonged ATP application the opening of a permeation pathway for large molecules is induced
We describe the identification and characterization of a novel P2X7 splice variant, P2X7(k), with an alternative N terminus and type; membrane domains (TMDs) 1

Summary

A Functional P2X7 Splice Variant with an Alternative

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. (23, 25) while the GlaxoϪ/Ϫ mice established its role in inflammatory and neuropathic pain [26] All these findings and multiple subsequent studies based on these mouse models defined the P2X7R as a promising target for the development of innovative therapeutic strategies, and selective P2X7 inhibitors are already in clinical trials for the treatment of rheumatoid arthritis [27]. Due to a novel alternative exon 1 and translation start, this splice variant escapes inactivation in the Glaxo P2X7Ϫ/Ϫ mice and could account for possible inconsistencies between results obtained with different P2X7Ϫ/Ϫ mouse lines [28]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION