Functional modulation of P(2x2) 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 P(2x2) 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 P(2x2) 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 P(2x2) 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 P(2x2) 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 P(2x2) 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 P(2x2) receptor and ATP-mediated physiological effects in the nervous system.