K(IR)2.1 INWARD RECTIFIER K+ CHANNELS ARE REGULATED INDEPENDENTLY BY PROTEIN-KINASES AND ATP HYDROLYSIS

Second messenger regulation of IRK1 (K(ir)2.1) inward rectifier K+ channels was investigated in giant inside-out patches from Xenopus oocytes. K(ir)2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with >10 mu M free Mg2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATP gamma S, this suggests an ATPase-like mechanism. In addition to ATP, the catalytic subunit of cAMP-dependent protein kinase (PKA) induced an increase in current amplitude, which could, however, only be observed if channels were previously or subsequently stimulated by Mg-ATP and free Mg2+. This indicates that functional activity of K(ir)2.1 channels requires both phosphorylation by PKA and ATP hydrolysis. Moreover, currents could be down-regulated by N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific stimulator of protein kinase C (PKC), suggesting that PKA and PKC mediate inverse effects on K(ir)2.1 channels. Regulation of K(ir)2.1 channels described here may be an important mechanism for regulation of excitability.