Identification of Thr283 as a key determinant of P2X7 receptor function.
OBJECTIVE
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.


METHODS
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)).


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.
Young
M T
MT
Department of Biomedical Science, University of Sheffield, Florey Building, Western Bank, Sheffield, UK.
Pelegrin
P
P
Surprenant
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013912:Threonine
C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D013912:Threonine; D000255:Adenosine Triphosphate; D002118:Calcium