Protein kinase C-dependent inhibition of Kir channels

Kir 通道的蛋白激酶 C 依赖性抑制

• 批准号: 6752128
• 负责人: Diomedes E. Logothetis
• 金额: $ 4.14万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752128
• 关键词: CHO cells; Xenopus oocyte; binding proteins; biological signal transduction; confocal scanning microscopy; electrophysiology; hydrolysis; kinase inhibitor; muscarine; phosphatidylinositols; polymerase chain reaction; potassium channel; protein isoforms; protein kinase C; protein protein interaction; tissue /cell culture; western blottings

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.

描述（由申请人提供） 最近人们认为，内部整流K+（KIR）通道家族成员的共同特征是，它们都被凤凰糖苷 - 磷酸 - 磷酸（PIP2）激活。特定的KIR成员之间已经描述了通道PIP2相互作用的差异，包括生物化学（Huang等，1998）和功能（Huang等，1998; Zhang等，1999; Lopes等，2002）。似乎有KIR通道表现出相对较强，中间或弱与PIP2的相互作用。 PIP2水解抑制了某些KIR通道。 Channei-PIP2相互作用的强度与通道活性的程度以及导致PIP2水解的信号引起的通道抑制程度相关。因此，与PIP2相互作用弱相互作用的通道受PIP2水解抑制最大，而与PIP2相互作用的通道并未受到PIP2水解的抑制。 PKC的通道调制以取决于通道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，强化通道PIP2相互作用的突变可以减弱或废除PKC的影响。同样，在与PIP2强烈相互作用并因此缺乏PKC依赖性抑制的通道中，削弱Channei-PIP2相互作用的突变可以使活化的PKC抑制通道。 PKC介导的抑制作用可以在异源系统中（例如在Xenopus卵母细胞）中获得，但在其他卵母细胞（例如Cho哺乳动物细胞）中不能获得。当前的提案旨在找到以下两个主要问题的答案。 （a）是否需要在不存在效果的某些细胞系统中重建PKC介导的电流抑制所需的特定PKC同工型？是否存在介导PKC效应的特定PKC衔接蛋白？ （b）由于PKC介导的效应而导致的毒蕈碱诱导的KIR电流诱导的抑制是什么？ KIR通道活性的PKC是否会削弱通道PIP2的相互作用？当前的提案扩大并增强了父母赠款HL59949。在父授予中，我们的重点是确定通道蛋白本身或相关蛋白质上的PKC依赖性磷酸化位点，并研究它们与Channei-Pipz相互作用位点的关系。在当前赠款中，重点是确定参与PKC介导的抑制作用的特定PKC同工型和/或衔接子蛋白，并确定PKC对ACH诱导的电流抑制的相对贡献与由于PIP2水解导致的直接通道PIP2相互作用的降低。