Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase.
It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.
Chow
Y W
YW
Department of Physiology, Chang Gung University School of Medicine, Tao-Yuan, Taiwan, Republic of China.
Wang
H L
HL
D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D012694:Serine / Q000378:metabolism
C546401:P2RX2 protein, human; C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C