Modulation of inwardly rectifying ATP-regulated K+ channel by phosphorylation process in opossum kidney cells.
The role of phosphorylation in modulating an inwardly rectifying ATP-regulated K+ channel with inward conductance of about 90 pS was examined using the patch-clamp technique on opossum kidney (OK) cells. The activity of the inwardly rectifying K+ channel observed in cell-attached patches rapidly declined (channel "rundown") upon excision of the membrane into inside-out patches in a control bath solution (3 mM Mg2+, ATP-free). The declined channel activity was partially restored by applying ATP to the bath, and the ATP-induced channel restoration reached the near maximal level at an ATP concentration of 3 mM. The channel activity maintained by 3 mM ATP in inside-out patches was inhibited by K-252a (10 microM), a nonspecific protein kinase inhibitor, or KT5720 (200 nm), a specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), and was further stimulated by the addition of a catalytic subunit of PKA (20 nM). In cell-attached patches, the channel activity was also inhibited by K-252a (10 microM) or KT5720 (200 nM). The application of dibutyryl-cAMP (100 microM) alone failed to enhance channel activity, but significantly stimulated channel activity after the pretreatment of cells with Ro-20-1724 (100 microM), an inhibitor of cAMP-specific phosphodiesterase. These results suggest that maintenance of the activity of ATP-regulated K+ channels in OK cells requires protein kinase-mediated phosphorylation with ATP-hydrolysis, and that phosphorylation is mainly induced by PKA.
Kubokawa
M
M
Department of Physiology, Osaka Medical College, Takatsuki, Japan.
Mori
Y
Y
Kubota
T
T
D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D026121:Indole Alkaloids; D007211:Indoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009893:Opossums; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D047428:Protein Kinase Inhibitors; D011758:Pyrroles / Q000494:pharmacology
D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007211:Indoles; D010726:Phosphodiesterase Inhibitors; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; C057416:KT 5720; D003994:Bucladesine; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D017868:Cyclic AMP-Dependent Protein Kinases