Regulation of large calcium-activated potassium channels by protein phosphatase 2A.
Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch-clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for 20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.
Sansom
S C
SC
Division of Renal Diseases and Hypertension and the Department of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School, Houston, Texas 77073, USA.
Stockand
J D
JD
Hall
D
D
Williams
B
B
D002448:Cell Adhesion; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D054648:Protein Phosphatase 2; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D019319:Okadaic Acid; D003995:Dibutyryl Cyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D006152:Cyclic GMP