Alioua A, Huggins J P, Rousseau E
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Am J Physiol. 1995 Jun;268(6 Pt 1):L1057-63. doi: 10.1152/ajplung.1995.268.6.L1057.
Modulation of Ca(2+)-dependent K+ channel (GKCa) activities in airway smooth muscles (ASM) by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) is thought to play a central role in mediating the effect of some bronchodilator agents that elevate cytoplasmic basal cGMP concentrations. However, no direct evidence supports this hypothesis in ASM. In the present work, we provide evidence that PKG-I alpha upregulates GKCa channels derived from bovine tracheal smooth muscle cells and reconstituted into planar lipid bilayers. In two different experimental approaches, PKG increased the open probability as well as the mean open time of GKCa channels, without any effect on unitary current amplitudes and unit conductance. Our results indicate that the kinetics of GKCa channels are controlled by a phosphorylation step mediated by PKG, and thus might be modulated by intracellular cGMP. Biochemical assays demonstrated that PKG phosphorylates several protein bands in the membrane fraction. Two of those proteins co-migrate with the same relative molecular mass as the 62- and 30-kDa components of the purified channel complex, identified as GKCa-alpha and -beta subunits, respectively. Our results also indicate that PKG phosphorylates the GKCa-alpha subunit with an apparent stoichiometry of 0.89, which would be consistent with the presence of a single PKG-sensitive phosphorylating site within its amino acid sequence. Furthermore, these results demonstrate for the first time that PKG directly phosphorylates GKCa from airway smooth muscle cells and thereby activates the channels at negative voltage or at low free Ca2+ concentrations.
鸟苷 3',5'-环磷酸（cGMP）依赖性蛋白激酶（PKG）对气道平滑肌（ASM）中钙依赖性钾通道（GKCa）活性的调节，被认为在介导某些能提高细胞质基础 cGMP 浓度的支气管扩张剂的作用中起核心作用。然而，在气道平滑肌中尚无直接证据支持这一假说。在本研究中，我们提供证据表明，PKG-Iα 上调源自牛气管平滑肌细胞并重组到平面脂质双分子层中的 GKCa 通道。在两种不同的实验方法中，PKG 增加了 GKCa 通道的开放概率以及平均开放时间，而对单通道电流幅度和单位电导没有任何影响。我们的结果表明，GKCa 通道的动力学受 PKG 介导的磷酸化步骤控制，因此可能受细胞内 cGMP 调节。生化分析表明，PKG 使膜组分中的几条蛋白带发生磷酸化。其中两种蛋白与纯化通道复合物的 62 kDa 和 30 kDa 组分具有相同的相对分子质量，分别被鉴定为 GKCa-α 和 -β 亚基。我们的结果还表明，PKG 以 0.89 的表观化学计量比使 GKCa-α 亚基磷酸化，这与在其氨基酸序列中存在单个 PKG 敏感的磷酸化位点一致。此外，这些结果首次证明 PKG 直接使气道平滑肌细胞中的 GKCa 磷酸化，从而在负电压或低游离 Ca2+ 浓度下激活通道。
