A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels

P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP rec eptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and volta ge clamp recording in Xenopus oocytes, we identified the highly conserved p rotein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (ti me constant, >1 min) rat P2X(2) receptors, Mutant receptors P2X(2)T18A, T18 N, and K20T devoid of this consensus site exhibited quickly desensitizing p roperties (time constant, <1 s), In contrast with wild-type receptors, muta nt P2X(2) receptors with truncated C terminus exhibited variable cell-speci fic kinetics with quickly desensitizing currents converted to slowly desens itizing currents by phorbol ester-mediated stimulation of protein kinase C, Phosphorylation of Thr(18) was demonstrated directly by immunodetection us ing specific monoclonal antibodies directed against the phosphothreonine-pr oline motif, Our data indicate that both phosphorylation of the conserved t hreonine residue in the N-terminal domain by protein kinase C and interacti on between the two cytoplasmic domains of P2X(2) subunits are necessary for the full expression of slowly desensitizing ATP-gated channels.