Large conductance voltage- and Ca2+-activated potassium stations (BK stations) are essential responses regulators in excitable cells and so are potently controlled by proteins kinases. BK route activity. We further recognized that proteins phosphatase 1 can be from the route continuously counteracting phosphorylation of S695. PKC phosphorylation at S1151 also affects excitement of BK route activity by proteins kinase G (PKG) CCT128930 and proteins kinase CCT128930 A (PKA). Although S1151A mutant route is triggered by PKA just CCT128930 the phosphorylation of S1151 by PKC makes the route attentive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or presenting a phosphomimetic aspartate as of this placement (S695D) 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. and = 7). Reduced Gmax could not be reversed by increasing voltage or Ca2+. The inhibitory PKC effect was abolished when inside-out patches were concurrently superfused for at least 5 min with PKCc and 5 μM of the PKC pseudosubstrate inhibitor peptide PKC19-31 (Fig. 1= 9) and prolonged the closed time from 23.9 ± 3.8 to 47.5 ± 4.7 ms (= 7). The single-channel conductance the voltage dependence and the calcium sensitivity of BK channels were not affected by PKC (Fig. S1). Fig. 1. PKC inhibits BK channel activity by decreasing channel open state probability. (= 15) and in the presence of 30 … CCT128930 PKC-Induced Inhibition Depends on Phosphorylation of Ser695 and Ser1151. By screening the BK channel α subunit for PKC consensus sequences (13) we found in addition to the prominent tandem motif with Ser1151 and Ser1154 seven further serines-Ser390 Ser499 Ser625 Ser630 Ser695 Ser712 and Ser1129-as putative PKC phosphorylation sites. Whole-cell with Fig. 1with Fig. 1= 6) which is 47.4 ??2.1% of the conductance measured in the nonmutated BK channel at the same potential. The mutant was insensitive toward PKC; a V1/2 of 47.9 ± 3.6 mV (= 6) vs. 51.6 ± 4.6 mV in seven controls indicates that its voltage-dependent activation was unchanged. Nearly identical results as those shown in Fig. 2were obtained with the double mutant S695D/S1151D. To determine the contribution of Ser1151 to the inhibitory effect of PKC we created the double mutant S695D/S1151A. This mutant showed exactly the same electrophysiological characteristics as the mutants S695D and S695D/S1151D its membrane conductance was strongly reduced with respect to the nonmutated BK channel (by 59.8 ± 3.9% CCT128930 at +80 mV; = 8) and PKC was ineffective (Fig. 2and and and = 12; V1/2 after PKG 25.1 ± 4.4 mV; = 6). Opposite to PKG the catalytic subunit of PKA (300 CCT128930 nM) had no significant influence on the S1151D mutant (V1/2 before 51.4 ± 4.2 mV = 12; V1/2 after PKA 48.5 ± 3.8 mV; = 6; Fig. 4= 14) and 50.7 ± 5.8 mV after the application of PKG (= 7; not significant). In contrast PKA shifted V1/2 by 29 mV from 52.1 ± 5.1 (= 14) to 22.7 ± 4.2 mV (= 7; Fig. 4and were obtained when Ser1151 was mutated on Rabbit Polyclonal to IKK-gamma (phospho-Ser31). the S695A background (Fig. S2). Because phosphorylation/dephosphorylation of Ser1151 seems to determine whether BK channels are regulated by PKG or PKA we wondered whether BK channels under the influence of 30 nM PKCc are still regulated by the two cyclic nucleotide-dependent kinases. When in the presence of PKCc PKG or PKA was applied to excised inside-out patches expressing the nonmutated BK channel neither kinase enhanced membrane conductance at any potential (Fig. 4 and = 6) and the double mutants S695D/S1151D (= 7) and S695D/S1151A (Fig. 4= 8). Mutation of Ser695 in alanine abolished the inhibitory PKC effect on BK channels but had no influence on the stimulatory effect of additionally applied PKG (Fig. S3). Thus BK channels in which Ser695 is phosphorylated by PKC or contain a negatively charged amino acid at this position are no longer regulated by cyclic nucleotide-dependent protein kinases. Fig. 4. Phosphorylation of serines 695 and 1151 determines the sensitivity of BK channels to PKA and PKG. (and = 16). Additional application of the membrane permeant-specific activators of PKG (300 μM.