C-terminal calmodulin-binding motif differentially controls human and rat P2X7 receptor current facilitation.
P2X(7) receptors (P2X(7)R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X(7)Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X(7)R resulted from a Ca(2+)-calmodulin-dependent process and a distinct calcium-independent process. However, P2X(7)Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X(7)R as well as rat/human, human/rat chimeric, and mutated P2X(7)Rs. Facilitation at the human P2X(7)R was 5-fold slower than at the rat P2X(7)R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X(7)R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X(7)R. Replacement of three critical residues of this binding domain from the rat into the human P2X(7)R (T541I, C552S, and G559V) reconstituted the Ca(2+)-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X(7)Rs. The absence of Ca(2+)-dependent facilitation at the human P2X(7)R may represent a protective adaptation of the innate immune response in which P2X(7)R plays significant roles.
Roger
Sébastien
S
Inserm U921, Université François Rabelais de Tours, 10 Boulevard Tonnellé, 37032 Tours, France. sebastien.roger@univ-tours.fr
Gillet
Ludovic
L
Baroja-Mazo
Alberto
A
Surprenant
Annmarie
A
Pelegrin
Pablo
P
D020816:Amino Acid Motifs; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058486:Receptors, Purinergic P2X7; D014966:Xanthenes / Q000494:pharmacology
D000814:Aniline Compounds; D002147:Calmodulin; C409648:Fluo 4; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D014966:Xanthenes; D002118:Calcium