Pei Z M, Ward J M, Harper J F, Schroeder J I
Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0116, USA.
EMBO J. 1996 Dec 2;15(23):6564-74.
Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.
高等植物中的钙依赖蛋白激酶（CDPKs）含有一个C端类钙调蛋白调节结构域。关于生理状态下CDPK的作用靶点知之甚少。激酶活性和多个Ca2+依赖的信号通路都与气孔保卫细胞运动的控制有关。为了确定CDPK或其他蛋白激酶是否在保卫细胞信号传导中发挥作用，在膜片钳实验期间，将纯化的和重组的激酶应用于蚕豆保卫细胞液泡。CDPK激活了保卫细胞中新型的液泡氯（VCL）和苹果酸电导。激活依赖于Ca2+和ATP。此外，使用不依赖Ca2+的组成型活性CDPK*突变体，在没有Ca2+的情况下也发生了VCL激活。蛋白激酶A的激活作用较弱（与CDPK相比为22%）。全液泡记录中的电流反转随Cl-的能斯特电位而变化，并在谷氨酸中消失。单通道记录显示有一个CDPK激活的34±5 pS Cl-通道。VCL通道在生理电位下被激活，使Cl-能够摄取到液泡中。VCL通道可能为气孔开放所需的阴离子摄取到液泡中提供了一个以前未被识别但必要的途径。在红甜菜液泡中也观察到了CDPK激活的VCL电流，这表明这些通道可能为激酶依赖的阴离子摄取提供了一种更普遍的机制。
