REGULATION OF LARGE CALCIUM-ACTIVATED POTASSIUM CHANNELS BY PROTEIN PHOSPHATASE 2A

Vasodilating agents induce relaxation of mesangial cells, in part thro ugh cGMP-mediated activation of large calcium-activated potassium chan nels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cG MP (Bt(2)cGMP) is characterized by a biphasic increase and then decrea se (''rundown'') in open probability. Using the patch clamp method in conjunction with phosphatase inhibitors, we investigated whether the r un-down phase was the result of dephosphorylation by an endogenous pro tein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhi bitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentr ations, caused a significantly greater and sustained response of BKCa to Bt(2)cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt(2)cGMP was greater than the response to these agents added separately, Incubation of mesangial cells with okadaic a cid for 20 min at a concentration (5 nM) specific for PP2A increased t he basal open probability of BKCa and completely inhibited rundown aft er activation by Bt(2)cGMP. Incubation with calyculin A (10 nM), a mor e potent inhibitor of PP1, did not affect BKCa activity, In inside-out patches, Bt(2)cGMP plus MgATP caused a sustained activation of BKCa t hat was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common subs trate for endogenous cGMP-activated protein kinase, which activates BK Ca, and PP2A, which inactivates BKCa, in human mesangial cells.