Conditional and Unconditional Inhibition of Calcium-activated Potassium Channels by Reversible Protein Phosphorylation

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, 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. 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.Large conductance, calcium-activated potassium channels (BK Ca 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). The sensitivity of BK Ca 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). Such reversible protein phosphorylation is the primary mechanism for regulating ion channel activity on the physiological time scale of seconds and minutes (2-4). Modification of the behavior of BK Ca channels has profound effects on the frequency and duration of action potentials in excitable cells, thereby controlling the physiological function of these cells.Electrophysiological measurements indicate that the activity of BK Ca channels in native cells may be up-or down-regulated by reversible protein phosphorylation. For example, protein kinase-induced phosphorylation enhances calcium-activated potassium currents in smooth muscle (5, 6), whereas in photoreceptors (7), hippocampal neurones (8 -11), and neurendocrine cells (12), the currents are inhibited by kinase activity. BK Ca channels exist as multimeric protein complexes composed of two integral membrane subunits, the pore-forming ␣ subunit and the regulatory ␤ subunit (1). All ␣ subunits are apparently coded by the same gene, the slo gene, but alternative RNA splicing during development produces functionally distinct channel proteins in different cell types (13-15). Hence, opposite modulatory effects ...

Dual role of protein kinase C on BK channel regulation

“…Note, however, that the phosphorylation of S3 STREX by PKA produced a ∼20-mV rightward shift of the voltage-dependent activation curve (25), whereas PKC reduced the maximal BK channel activity without affecting the apparent voltage sensitivity. A similar observation has been made earlier with PKC on BK channels in pituitary CH 4 C 1 cells (26).…”

“…Similar to the results obtained with the mutant BK channel S 1151 A (Fig. 4B), PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] prevented the stimulatory action of PKG in TSMCs and switched the responsiveness of the channel toward PKA. At a holding potential of 40 mV, NP o was not significantly changed by PKG (control NP o 0.44 ± 0.05; after PKG 0.37 ± 0.05, n = 6), whereas a 2.4-fold increase was induced by PKA (control NP o 0.47 ± 0.03; after PKA 1.19 ± 0.17, n = 6; Fig.…”

“…Reduced G max could not be reversed by increasing voltage or Ca 2+ . The inhibitory PKC effect was abolished when inside-out patches were concurrently superfused for at least 5 min with PKC c and 5 μM of the PKC pseudosubstrate inhibitor peptide PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] (Fig. 1A).…”

“…To allow complete dephosphorylation of PKC sites, inside-out patches were first superfused for 5 min with ATP-free solution to which 5 μM PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] had been added. This solution, which did not significantly alter NP o , was then changed for another 5 min to a solution containing ATP, PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] , and either PKG or PKA. Similar to the results obtained with the mutant BK channel S 1151 A (Fig.…”

Resting Potential and Submembrane Calcium Concentration of Inner Hair Cells in the Isolated Mouse Cochlea Are Set by KCNQ-Type Potassium Channels

“…In this study, we used BK channels as sensors to estimate [Ca 2ϩ (Cui et al, 1997). Although all BK channels are transcribed from a single gene locus, Slo, their Ca 2ϩ sensitivity differs between cell types because of extensive alternative splicing (Tseng-Crank et al, 1994), association with ␤-subunits (McManus et al, 1995; Brenner et al, 2000), and protein phosphorylation (Levitan, 1994;Hall and Armstrong, 2000). We therefore determined activation parameters in inside-out patches from IHCs using different [Ca 2ϩ ] i .…”