Abstract
Large conductance, calcium-activated potassium channels (BK(Ca) or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK(Ca) channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK(Ca) 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 BK(Ca) 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 BK(Ca) channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.
Highlights
Large conductance, calcium-activated potassium channels (BKCa or maxi-K channels) are uniquely powerful determinants of electrical activity in the nervous, endocrine, and vascular systems, since they respond directly to both membrane depolarization and calcium accumulation (1)
Dephosphorylated BKCa Channels Respond to Voltage and Calcium in the Physiological Ranges—Once BKCa channel activity had stabilized in MgATP-free solution, it remained stable for the duration of the recording
The data reported here establish clearly that native calciumactivated potassium channels with the largest conductance (BKCa or maxi-K) are inhibited by endogenous protein kinases and stimulated by endogenous protein phosphatases that remain closely associated with the channels in cell-free patches from an immortalized endocrine rat pituitary cell line (GH4C1)
Summary
3749 –3754, 2000 Printed in U.S.A. Conditional and Unconditional Inhibition of Calcium-activated Potassium Channels by Reversible Protein Phosphorylation*. Calcium-activated potassium channels (BKCa or maxi-K) are important determinants of membrane excitability in many cell types. Endogenous protein kinases inhibited BKCa channel activity at two functionally distinct sites. A closely associated, cAMPdependent 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. Conditional versus unconditional inhibition of BKCa channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability. The sensitivity of BKCa channels to such physiological stimuli can be modified by enzyme pathways mediating reversible phosphorylation of the ion channel proteins or other closely associated regulatory proteins (2). Modification of the behavior of BKCa channels has profound effects on the frequency and duration of action potentials in excitable cells, thereby controlling the physiological function of these cells
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