Protein kinase C-dependent inhibition of Kir channels

描述(由申请人提供) 最近人们认识到，内向整流钾(KIR)通道家族成员的一个共同特征是它们都被磷脂酰肌醇-二磷酸(PIP2)激活。已经描述了特定KIR成员之间的通道-PIP2相互作用的差异，既有生化上的(Huang等人，1998)，也有功能上的(Huang等人，1998；Zhang等人，1999；Lope等人，2002)。似乎存在KIR通道，显示出与PIP2相对较强、中等或较弱的相互作用。某些KIR通道被PIP2水解酶抑制。ChannI-PIP2相互作用的强度与通道活动的程度和导致PIP2水解的信号引起的通道抑制程度相关。因此，与PIP2相互作用弱的通道受到PIP2水解的抑制最多，而与PIP2相互作用强烈的通道不受PIP2水解的抑制。由PKC进行的通道调制以依赖于通道-PIP2相互作用的方式影响通道活动。因此，在显示PKC介导的活性抑制的野生型通道中，加强通道-PIP2相互作用的突变可以减弱或取消PKC的作用。同样，在与PIP2强烈相互作用的通道中，因此缺乏PKC依赖的抑制，减弱ChannI-PIP2相互作用的突变可以使通道可被激活的PKC抑制。PKC介导的抑制作用可以在异源系统中获得，例如在非洲爪哇卵母细胞中，而在其他系统中，如CHO哺乳动物细胞中则不能。目前的提案旨在找到以下两个主要问题的答案。(A)在某些细胞系统中，是否有特定的PKC亚型需要重建PKC介导的电流抑制，而这种抑制作用是不存在的？是否有特定的PKC接头蛋白参与介导PKC效应？(B)由于PKC介导的效应，毒扁豆碱对KIR电流的抑制程度有多大？KIR通道活性的PKC调节是否削弱了通道-PIP2的相互作用？目前的提案扩大和加强了家长补助金HL59949。在父母的资助中，我们的重点是确定通道蛋白本身或相关蛋白上依赖于PKC的磷酸化位点，并研究它们与ChannI-PIPz相互作用位点的关系。在目前的GRANT中，重点是确定参与PKC介导的抑制的特定PKC亚型和/或适配蛋白，并确定PKC对ACh诱导的电流抑制的相对贡献，以及PIP2水解导致的直接通道-PIP2相互作用的减少。

DESCRIPTION (provided by applicant) It has been recently appreciated that a common characteristic of members of the inwardly rectifying K+ (Kir) channel family is that they are all activated by phospatidylinositol-bis-phosphate (PIP2). Differences in channel-PIP2 interactions have been described among specific Kir members, both biochemically (Huang et al., 1998) and functionally (Huang et al., 1998; Zhang et al., 1999; Lopes et al., 2002). There seem to be Kir channels that show either relatively strong, intermediate or weak interactions with PIP2. Certain Kir channels are inhibited by PIP2 hydrolysis. The strength of channeI-PIP2 interactions correlates with the extent of channel activity and the degree of channel inhibition caused by signals that lead to PIP2 hydrolysis. Thus, channels that interact weakly with PIP2 are inhibited the most by PIP2 hydrolysis, while channels that interact strongly with PIP2 are not inhibited by PIP2 hydrolysis. Channel modulation by PKC affects channel activity in a manner dependent on channel-PIP2 interactions. Thus, in a wild-type channel showing PKC-mediated inhibition of activity, mutations strengthening channel-PIP2 interactions can attenuate or abolish the effect of PKC. Similarly, in a channel that interacts strongly with PIP2 and therefore lacks PKC-dependent inhibition, mutations that weaken channeI-PIP2 interactions can render the channel inhibitable by activated PKC. The PKC-mediated inhibition could be obtained in heterologous systems such as in Xenopus oocytes but not in others, such as the CHO mammalian cells. The current proposal aims to find answers to the following two major questions. (a) Are there specific PKC isoforms that are needed to reconstitute the PKC-mediated current inhibition in certain cell systems where the effect is absent? Are there specific PKC adaptor proteins involved in mediating the PKC effects? (b) To what extend is the muscarinic induced inhibition of Kir currents due to PKC mediated effects? Does PKC modulation of Kir channel activity weaken channel-PIP2 interactions? The current proposal expands and enhances the parent grant HL59949. In the parent grant our emphasis is to identify the PKC-dependent phosphorylation sites either on the channel protein itself or associated proteins and to study their relationship to channeI-PIPz interacting sites. In the current grant the focus is to identify the specific PKC isoforms and/or adaptor proteins involved in the PKC-mediated inhibition and to determine the relative contribution to the ACh-induced current inhibition by PKC versus the decrease in direct channel-PIP2 interactions that result due to the PIP2 hydrolysis.