Structural Determinants of PIP2 Regulation

 描述（由申请人提供）：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，可能导致危及生命的心律失常。PIP 2是一种信号磷脂，在过去的二十年中被认为是离子通道活性的主要调节剂。虽然我们已经了解了关于PIP 2如何引起+通道门控的控制细胞静息电位的内向整流K（Kir）通道，但我们对控制动作电位的+复极化相的电压门控K（Kv）通道中类似的PIP 2控制的理解是缺乏的。这项提议旨在将我们对这两类重要离子通道的理解提升到一个新的水平。对于Kir通道，具有与PIP 2复合的Kir3.2的晶体结构和我们产生全长纯化蛋白的能力，我们准备好探索通过蛋白磷酸化控制活性的机制。在过去的十年中，我们已经认识到通过特异性激酶的Kir 3通道磷酸化可以分别通过增强或延迟对PIP 2的敏感性来刺激（例如蛋白激酶A-PKA）或抑制（例如蛋白激酶C-PKC）活性。在这个提议中，我们假设PKC对特定残基的策略性磷酸化与PIP 2竞争带正电荷的Kir 3通道-PIP 2相互作用残基，从而削弱通道协调PIP 2的能力并导致通道活性的抑制。我们建议测试这一假设，通过鉴定在体外PKC磷酸化的残基Kir 3通道，使用质谱和采用计算建模，诱变和电生理学来探测的机制，其中一个特定的残基的磷酸化改变通道PIP 2的相互作用，抑制通道活性。对于KV通道，我们提出了强有力的初步结果表明，PIP 2控制Kv2.1通道缓慢失活，像其他（电压依赖性和电压不依赖性）通道导致崩溃的选择性过滤器。这些新的数据使我们假设Kv2.1 PIP 2相互作用残基通过孔衬S6螺旋中间的中继残基与选择性过滤器连接。我们建议使用计算模型，诱变和电生理学来测试这一假设，并建立这种新的机制来控制Kv通道的活性，这可能会在显示缓慢失活的其他Kv通道中起作用。本基金第5个周期的研究结果将促进我们对PIP 2门控th及其通过翻译后修饰机制调节通道活性的理解。我们从Kir 3通道的结构见解将有利于我们识别新的PIP 2敏感的Kv2.1通道残基。我们对Kv2.1 PIP 2相互作用残基与该通道的选择性过滤器耦合的新见解肯定会指导我们在Kir 3通道中进行长期研究，以比较PIP 2相互作用位点与这些通道的选择性过滤器的耦合，这是Kir领域尚不清楚的过程。

 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.