Protein Phosphatase 2A Is Essential for the Activation of Ca²⁺-activated K⁺ Currents by cGMP-dependent Protein Kinase in Tracheal Smooth Muscle and Chinese Hamster Ovary Cells*

The regulation of Ca²⁺-activated K⁺ channels (K_Ca channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Iα (CHO-cGK cells), K_Ca channels with intermediate conductance (∼50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca²⁺-activated K⁺ currents had a higher sensitivity toward the cytosolic Ca²⁺ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 nM) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca²⁺-activated K⁺ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NP_o) of maxi-K_Ca channels (conductance of ∼260 picosiemens) was enhanced by 300 µM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 µM isoprenaline, 20 µM forskolin, and 3 mM 8-bromo-cAMP failed to enhance K_Ca channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NP_o when applied to the cytosolic side of the patch. The enhancement of NP_o by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 µM) and the protein phosphatase (PP) inhibitors microcystin (5 µM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NP_o in excised inside-out patches. The results show that cGMP kinase regulates two different K_Ca channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the K_Ca channel is specific for cGMP kinase and is not mimicked by cAMP kinase.