Regulation of inwardly rectifying K+ channel in cultured opossum proximal tubule cells by protein phosphatases 1 and 2A

The inwardly rectifying ATP-regulated K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cells is activated at least in part by protein kinase A (PKA) , In this study, we examined the effects of protein serine/threonine phosph atase types 1 (PP-1) and 2A (PP-2A) on activity of the K+ channel using the patch-clamp technique. In cell-attached patches, channel activity was enha nced by the application of okadaic acid (OA, 1 mu M), a membrane-permeable inhibitor of PP-I and PP-2A, to the bath solution. This enhancement was abo lished by the pretreatment of cells with KT5720 (200 nM), a specific inhibi tor of PKA, In inside-out patches, channel activity which could be maintain ed in the presence of ATP (3 mM) in the bath solution was also increased by the addition of OA (1 mu M), and the OA-induced increase in channel activi ty was partially prevented in the presence of KT5720 (200 nM), Direct appli cation of either PP-I (1 U/ml) or PP-2A (1 U/ml) to the cytoplasmic surface of the patch membrane inhibited channel activity maintained by ATP (3 mM) in inside-out patches. Moreover, channel activity stimulated by PKA (20 nM) in the presence of ATP (3 mM) was also inhibited by the application of eit her PP-1 (1 U/ml) or PP-2A (1 U/ml). These results indicate that the OA-sen sitive protein phosphatase is involved in the regulation of channel activit y, and suggest that both PP-1 and PP-2A are candidates responsible for the inhibition of channel activity through dephosphorylation of the PKA-mediate d protein phosphorylation.