Large conductance voltage-and Ca 2+ -activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S 695 by PKC, located between the two regulators of K + conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S 1151 in the C terminus of the channel α-subunit. Phosphorylation of only one α-subunit at S 1151 and S 695 within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S 695 . PKC phosphorylation at S 1151 also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S 1151 A mutant channel is activated by PKA only, the phosphorylation of S 1151 by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S 695 by PKC or introducing a phosphomimetic aspartate at this position (S 695 D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.phosphorylation | protein kinase A | protein kinase G | protein phosphatase 1 | tracheal smooth muscle cells L arge conductance Ca 2+ -activated potassium channels (BK channels) are unique in their regulation by both intracellular Ca 2+ and membrane voltage. They are expressed in many tissues, and are particularly abundant in nerve and smooth muscle, where they play a key role as negative and positive feedback regulators of cell excitability by conducting repolarizing and hyperpolarizing outward currents, as has been impressively demonstrated in mice with targeted deletion of the pore-forming α-subunit (1-5). Alternative splicing of premRNA and protein phoshorylation generates structural and functional diversity of BK channels. Several serine/ threonine kinases, such as the cAMP-(PKA)-and cGMPdependent protein kinase (PKG) and protein kinase C (PKC), potently regulate BK channel activity. Their phosphorylation sites at the pore-forming α-subunit are fully conserved in almost all mammalian alternative splice variants, and mutation of the PKA and PKG phosphorylation sites abolished the kinase effect on channel activity in heterologous expression systems (6-10). In smooth muscle, PKA and PKG predominantly activate BK channels by increasing the apparent voltage and Ca 2+ sensitivity of the channel, whereas PKC exerts opposite effects (11). Experimental evidence indicates that hormones and drugs that activate PKA or PKG contribute to smooth muscle relaxation by activation of BK channels. In contrast, activ...

Airway smooth muscle is richly endowed with muscarinic receptors of the M 2 and M 3 subtype. Stimulation of these receptors inhibits large conductance calcium-activated K ؉ (BK) channels, a negative feed back regulator, in a pertussis toxinsensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M 2 or M 3 receptor (M 2 R or M 3 R). In M 2 R-but not M 3 Rexpressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M 2 R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the G␤␥ scavenger transducin-␣. In inside-out patches, direct application of 300 nM purified G␤␥ decreased channel open probability by 55%. The physical interaction of G␤␥ with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (ϳ20%) extent. Mouse tracheal cells responded similarly to CCh, purified G␤␥ and phospholipase C/protein kinase C inhibition as M 2 R-expressing HEK293 cells. Our results demonstrate that airway M 2 Rs inhibit BK channels by a dual, G␤␥-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.The parasympathetic nerves provide the dominant autonomic control of airway smooth muscle (ASM).3 The neurotransmitter acetylcholine exerts its effects by binding to muscarinic receptors of which five subtypes (M 1 -M 5 ) have been identified. All are members of the heptahelical family of G protein-coupled receptors (1-3). On mammalian ASM, the M 2 (M 2 R) and M 3 (M 3 R) receptor subtypes are expressed with M 2 R representing the larger population (4, 5). Additionally, M 4 receptors (M 4 R) are present in rabbit small airways and parenchyma (4, 6), but their function remains unknown. M 3 Rs in ASM are coupled to phospholipase C (PLC)/protein kinase C (PKC) pathway via pertussis toxin (PTX)-insensitive G proteins of the G q/11 family. The contractile response evoked by M 3 R stimulation is attributed to the formation of inositol trisphosphate (IP 3 ), the subsequent release of Ca 2ϩ from intracellular stores, the additional influx of extracellular calcium, and the Ca 2ϩ -sensitizing effect of PKC (7-11). Stimulation of M 2 muscarinic receptors (M 2 Rs) in ASM inhibits adenylyl cyclase via activation of PTX-sensitive G proteins of the G i/o family (12, 13), and therefore M 2 Rs are thought to counteract relaxation that requires activation of adenylyl cyclase, as for example the ␤ 2 -adrenoreceptor agonist-induced ASM relaxation (14). Recently, however, evidence has been provided that M 2 Rs participate directly in ASM contraction. In mice with a targeted deletion of the M 2 R, the muscarinic receptor agonist carbamoylcholine (CCh) induced less ASM contraction than in the wild-type littermates (15). In addition,...