Fukao M, Mason H S, Britton F C, Kenyon J L, Horowitz B, Keef K D
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
J Biol Chem. 1999 Apr 16;274(16):10927-35. doi: 10.1074/jbc.274.16.10927.
NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.
一氧化氮（NO）诱导的环磷酸鸟苷（cGMP）依赖性蛋白激酶（PKG）激活增加了大电导钙激活钾通道的开放概率，并导致平滑肌舒张。然而，NO-PKG途径对通道调节的分子机制在克隆通道上尚未明确。本研究旨在阐明PKG在分子水平上对通道的调节作用。编码大电导钙激活钾通道α亚基（cslo-α）的cDNA在人胚肾293（HEK293）细胞中表达。cslo-α的全细胞和单通道特性表现出平滑肌细胞中天然大电导钙激活钾通道的功能特征。在全细胞记录中，NO供体硝普钠使外向电流增加了2.3倍。在细胞贴附式膜片中，硝普钠使cslo-α通道的开放概率（NPo）增加了3.3倍，而不影响单通道电导。PKG抑制剂KT5823抑制了硝普钠的刺激作用。仅在存在三磷酸腺苷（ATP）和cGMP的情况下，将PKG-Iα直接应用于内向外膜片的胞质表面，可使NPo增加3.2倍，而不影响单通道电导。将cslo-α中丝氨酸1072（PKG磷酸化的唯一最佳共有序列）被丙氨酸取代的点突变消除了PKG对内向外膜片中NPo的影响以及硝普钠对细胞贴附式膜片中的影响。这些结果表明，PKG通过对丝氨酸1072的直接磷酸化激活cslo-α。
