Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072
Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072
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DOI：
10.1074/jbc.274.16.10927
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发表时间：
1999-04-16
影响因子：
4.8
通讯作者：
Keef, KD
中科院分区：
文献类型：
--
作者：
Fukao, M;Mason, HS;Keef, KD
NO诱导的cGMP依赖性蛋白激酶（PKG）激活增加了大电导Ca 2+激活的K+通道的开放概率，导致平滑肌松弛，但NO-PKG途径调节通道的分子机制尚未在克隆通道上确定。本研究旨在阐明PKG介导的通道在分子水平上的调制。在HER 293细胞中表达编码大电导Ca 2+激活的K+通道的α亚基的cDNA，cslo-α。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Ca 2+激活的K+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2.3倍。在细胞贴附贴片中，硝普钠使cslo-α通道的通道开放概率（NPo）增加3.3倍，而不影响单位电导。硝普钠的刺激作用被PKG抑制剂KT 5823所抑制。直接应用PKG-I α的胞质表面的由内而外补丁增加NPO 3.2倍，只有在ATP和cGMP的存在下，不影响单一的电导。其中Ser-1072（PKG磷酸化的唯一最佳共有序列）被Ala取代的cslo-α的点突变消除了PKG对由内而外的斑块中的NPo的作用和SNP在细胞附着的斑块中的作用。这些结果表明PKG通过在丝氨酸1072处直接磷酸化来激活cslo-α。
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 HER293 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-I alpha 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.