Identification of Thr 283 as a key determinant of P2X 7 receptor function

Abstract

Background and purpose: The ATP-gated P2X 7 receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X 7 receptor mutations. Experimental approach: There are three differences in the sequences of P2X 7 cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe 11,Ala 221,Met 283]P2X 7 in the former and [Leu 11,Thr 221,Thr 283]P2X 7 in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu 451]P2X 7 vs [Pro 451]P2X 7). Key results: Maximum current densities at [Phe 11,Ala 221, Met 283]P2X 7 were <12% of those at [Leu 11,Thr 221,Thr 283]P2X 7 without change in the agonist concentration-response. Replacing methionine with threonine at residue 283 yielded a receptor whose properties were the same as [Leu 11,Thr 221,Thr 283]P2X 7. Replacing T283 in the rat P2X 7 receptor with methionine yielded currents that were <10% of wildtype and no ethidium uptake was associated with its activation. Maximum current densities and agonist EC 50 values were the same at mouse [Thr 283,Leu 451]P2X 7 and [Thr 283,Pro 451]P2X 7 but ethidium uptake and Fluo4 fluorescence were significantly reduced at the [Thr 283,Leu 451]P2X 7 receptor. There was equivalent surface membrane expression of all P2X 7 receptors. Conclusions: This study has revealed a residue (Thr 283) in the ectodomain that is critical for P2X 7 receptor function and suggests that the intracellular residue 451 alters downstream signalling independently of ion channel activity. © 2006 Nature Publishing Group All rights reserved.