Wang Z W, Kotlikoff M I
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Am J Physiol. 1996 Jul;271(1 Pt 1):L100-5. doi: 10.1152/ajplung.1996.271.1.L100.
The regulation of calcium-activated potassium (KCa) channels by endogenous protein kinase A (PKA) was examined in inside-out patches from equine tracheal myocytes. In the absence of exogenous protein kinases, ATP (500 microM) significantly augmented KCa channel activity when applied to the cytosolic patch surface [open-state probability (nP0, mean +/- SE) increased from 0.010 +/- 0.001 to 0.034 +/- 0.005 (n = 24)]. The stimulatory effect of ATP was mimicked by ATP-gamma-S but not by AMP-PNP. Rather, AMP-PNP significantly inhibited channel activity. The PKA inhibitory peptide (PKI) significantly reduced nPo and prevented the augmenting effect of subsequent ATP. Ht 31, an inhibitory peptide for A-kinase-anchoring proteins (AKAP), but not its proline-substituted mutant, also blocked the stimulatory effect of ATP. These results suggest that 1) ATP augments KCa channel activity through phosphorylation; 2) the phosphorylation is catalyzed by endogenous PKA; 3) anchoring via AKAP is required to maintain association of PKA with the membrane; and 4) in a newly obtained patch, some of the KCa channels are probably already in a phosphorylated state.
在马气管肌细胞的内面向外膜片中研究了内源性蛋白激酶A（PKA）对钙激活钾（KCa）通道的调节作用。在不存在外源性蛋白激酶的情况下，当将ATP（500 microM）施加于胞质膜片表面时，可显著增强KCa通道活性[开放状态概率（nP0，平均值±标准误）从0.010±0.001增加至0.034±0.005（n = 24）]。ATP的刺激作用可被ATP-γ-S模拟，但不能被AMP-PNP模拟。相反，AMP-PNP显著抑制通道活性。PKA抑制肽（PKI）显著降低nPo并阻止随后ATP的增强作用。Ht 31是一种A激酶锚定蛋白（AKAP）的抑制肽，但其脯氨酸取代突变体则不能，它也阻断了ATP的刺激作用。这些结果表明：1）ATP通过磷酸化增强KCa通道活性；2）磷酸化由内源性PKA催化；3）通过AKAP锚定是维持PKA与膜结合所必需的；4）在新获得的膜片中，一些KCa通道可能已经处于磷酸化状态。
