FUNCTIONALLY IMPORTANT PKA PHOSPHORYLATION SITE IN A KIR3 CHANNEL SUBUNIT

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CAMP依赖的蛋白激酶(PKA)使Kir3通道的活性增强，增强通道与PIP2的相互作用，而蛋白激酶C(PKC)的磷酸化则导致相反的作用。我们利用质谱仪鉴定了蛋白激酶处理后Kir3.1通道亚单位内的磷酸化位点。我们重点研究了Kir3.1C-末端的胞液结构域，据报道该结构域受几个调节器的调节。在体外，PKA的磷酸化在用磷蛋白染色处理时显示出令人信服的信号。磷酸化的C末端用MALDI-TOF/MS进行了质谱分析，质量位移为80U的多肽峰可能与磷酸基团的添加有关，然后经过串联MS(MS2和MS3)以确定修饰的位置。利用这种方法，我们确定了S385是一个体外磷酸化位点。该残基突变为丙氨酰残基，导致Kir3.1*电流对H89和forskolin的敏感性降低，这表明Kir3.1通道亚单位的这个新位点在PKA对其调节中起到了体内作用。 一篇描述这项工作的论文已经发表(Rusinova R，Shenym，Dolios G，Padovan J，Yang H，Kirchberger M，Wang R，Logothetis DE。质谱分析显示，在Kir3通道亚基中有一个重要的PKA磷酸化位点。弗卢格斯拱门。2009年6月；458(2)：303-14)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP2 interactions whereas phosphorylation by protein kinace C (PKC) leads to opposite effects. We utilized mass spectrometry to identify the phosphorylation sites within the Kir3.1 channel subunit upon treatment with protein kinases. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using MALDI-TOF/MS. Peptide peaks with a mass shift of 80u, which may relate to the addition of a phosphate group, were then subjected to tandem MS (MS2 and MS3) in order to determine the location of the modification. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to an alanyl residue resulted in a reduced sensitivity of Kir3.1* currents to H89 and forskolin, suggesting an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA. A paper describing this work has been published (Rusinova R, Shen YM, Dolios G, Padovan J, Yang H, Kirchberger M, Wang R, Logothetis DE. Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit. Pflugers Arch. 2009 Jun;458(2):303-14).