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 (BK(Ca)). Normally quiescent in cell-attached patches, the response of BK(Ca) 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 BK(Ca) to Bt₂cGMP. Within 2 min, the response of BK(Ca) 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 BK(Ca) and completely inhibited rundown after activation by Bt₂cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BK(Ca) activity. In inside-out patches, Bt₂cGMP plus MgATP caused a sustained activation of BK(Ca) that was inhibited by exogenous PP2A but not PP1. It is concluded that either BK(Ca) or a tightly associated regulator of BK(Ca) is a common substrate for endogenous cGMP-activated protein kinase, which activates BK(Ca), and PP2A, which inactivates BK(Ca), in human mesangial cells.