Conditional and unconditional inhibition of calcium-activated potassium channels by reversible protein phosphorylation

Large conductance, calcium-activated potassium channels (BKCa or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulatiom of native BKCa channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GE(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BKCa channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BKCa channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BKCa channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.