Structural Determinants of PIP2 Regulation

 说明(申请人提供)：钾(K+)通道是心脏电活动的关键决定因素，当它们发生故障时，可能会导致危及生命的心律失常。PIP2是一种信号磷脂，在过去的二十年里一直被认为是离子通道活性的主要调节因子。尽管我们已经在控制细胞静息电位的内向整流K(KIR)通道中了解了最多关于PIP2如何引起+通道门控的知识，但我们对控制动作电位+复极阶段的电压门控K(Kv)通道中类似的PIP2控制的了解还很少。这一建议旨在将我们对这两类重要离子通道的理解推向新的水平。对于KIR通道，由于具有Kir3.2 in与PIP2的复合体的晶体结构，以及我们生产全长纯化蛋白的能力，我们准备探索通过蛋白质磷酸化来控制活性的机制。在过去的十年里，我们已经认识到，特定的激酶对Kir3通道的磷酸化可以分别通过增强或抑制对PIP2的敏感性来刺激(如蛋白激酶A-PKA)或抑制(如蛋白激酶C-PKC)的活性。在这一建议中，我们假设PKC对特定残基的战略性磷酸化与PIP2竞争正电荷的Kir3通道-PIP2相互作用残基，从而削弱通道协调PIP2的能力，并导致通道活性的抑制。我们建议通过使用质谱学在体外鉴定Kir3通道的PKC磷酸化残基，并利用计算建模、突变和电生理学来探索特定残基的磷酸化改变通道-PIP2相互作用并抑制通道活性的机制来检验这一假说。对于Kv通道，我们提出了强有力的初步结果，表明PIP2控制着Kv2.1通道的缓慢失活，这与其他(电压依赖和电压无关)通道一样，会导致选择性过滤器的崩溃。这些新的数据导致我们假设Kv2.1 PIP2相互作用的残基通过孔衬S6螺旋中间的中继残基与选择性过滤器相连。我们建议使用计算模型、诱变和电生理学来验证这一假说，并建立这种控制Kv通道活动的新机制，该机制可能在其他表现出缓慢失活的Kv通道中工作。这项资助的5个周期的拟议研究结果将促进我们对PIP2门控TH及其通过翻译后修饰机制来调节通道活动的调节的理解。我们对Kir3通道的结构洞察将有助于我们识别新的对PIP2敏感的Kv2.1通道残基。我们对Kv2.1 PIP2相互作用残基与该通道选择性过滤器的耦合的新见解肯定将引导我们在Kir3通道中进行长期研究，以比较PIP2相互作用位点的耦合与这些通道的选择性过滤器，这一过程在KIR领域仍不清楚。

 DESCRIPTION (provided by applicant): Potassium (K+) channels are critical determinants of the electrical activity of the heart and when they malfunction life-threatening arrhythmias can result. PIP2 is a signaling phospholipid that for the past two decades has been recognized as a master regulator of ion channel activity. Although we have learned the most in inwardly rectifying K (Kir) channels that control the resting potential of cells about how PIP2 causes + channel gating, our understanding of similar PIP2 control in voltage-gated K (Kv) channels that control the + repolarization phase of the action potential is lacking. This proposal aims to push our understanding to the next level in each of these two important classes of ion channels. For Kir channels, with a crystal structure of Kir3.2 in complex with PIP2 and our ability to produce full-length purified protein we are ready to probe the mechanism of control of activity by protein phosphorylation. For the past decade we have realized that Kir3 channel phosphorylation by specific kinases can stimulate (e.g. Protein Kinase A - PKA) or inhibit (e.g. Protein Kinase C - PKC) activity by enhancing or retarding sensitivity to PIP2, respectively. In this proposal we hypothesize that the strategic phosphorylation of specific residues by PKC compete with PIP2 for positively charged Kir3 channel-PIP2 interacting residues, thus weakening the channel's ability to coordinate PIP2 and causing inhibition of channel activity. We propose to test this hypothesis by identifying in vitro PKC phosphorylated residues of Kir3 channels using Mass Spectrometry and employing computational modeling, mutagenesis and electrophysiology to probe the mechanism by which phosphorylation of a specific residue alters channel-PIP2 interactions and inhibits channel activity. For Kv channels, we present strong preliminary results showing that PIP2 controls the Kv2.1 channel slow inactivation which like other (voltage-dependent and voltage-independent) channels leads to a collapse of the selectivity filter. These novel data lead us to hypothesize that Kv2.1 PIP2 interacting residues are linked to the selectivity filter via a relay residue in the middle of the pore-lining S6 helix. We propose to tes this hypothesis using computational models, mutagenesis and electrophysiology and establishing this novel mechanism for controlling Kv channel activity, which may operate in additional Kv channels that display slow inactivation. Results from the proposed studies in the 5 cycle of this grant will advance our understanding of PIP2 gating th and its modulation by post-translational modification mechanisms to adjust channel activity. Our structural insights from Kir3 channels will be beneficial in our identification of novel PIP2-sensitive Kv2.1 channel residues. Our novel insights of the coupling of Kv2.1 PIP2 interacting residues to the selectivity filter of this channel will certainly guide us to pursue long-term studies in Kir3 channels to compare the coupling of PIP2- interacting sites to the selectivity filter of these channels, a process that remains unclear in the Kir field.