Structural Determinants of PIP2 Regulation

描述（由申请人提供）：信号磷脂PIP2最近因其在调节许多离子通道和转运蛋白活性中的关键作用而受到重视。PTP2如何控制如此多的跨膜蛋白的活性的分子细节尚不清楚。在阐明一些离子通道蛋白的三维结构方面的最新进展使得在原子水平上详细研究这个问题成为可能。在这里，我们建议确定由PIP2控制的K通道门控的两个端点：与PTP2的通道相互作用位点和它们控制的通道门。首先，我们将利用电生理学和诱变技术以及替代半胱氨酸接近性方法（SCAM）来探测PIP2在通道门控过程中的状态依赖性修饰，从而确定PIP2在内整流K （Kir）通道中控制的通道门。其次，我们将研究Na门控在Kir3和其他Kir通道中的变构效应，以及Kir通道- pip2相互作用的原子细节。这些研究将使我们能够理解这些相互作用的功能后果，如对PIP2的明显通道亲和力和与不同通道位点的磷酸肌苷立体特异性相互作用。在与本系计算生物学家的密切合作下，我们开发了一种结合电生理学和理论的方法来分析PIP2敏感通道：我们首先通过计算磷酸盐探针通道的分子相互作用场来定义它们的PIP2相互作用区域；利用互补的计算方法，我们使用磷酸肌苷探针运行布朗动力学（BD）模拟来定义PIP2相互作用残基。我们比较了这两种计算方法，它们的一致性证明了下一层次的分析，即通过分子动力学模拟对BD结构进行细化，以生成可以通过实验测试的可靠模型。这种结合实验/理论方法的有效性已经在Kir通道中得到了发展和加强，在那里我们已经产生了与PIP2相互作用相关的氨基酸残基的费力的实验证据。在目前这项工作的竞争性延续中，我们建议将这些研究扩展到电压门控K （Kv）通道家族，特别是因为新的Kv1.2晶体结构的可用性。我们提供的初步数据表明，不同的Kv亚家族成员对pip2敏感，并提出了与Kir通道相似的PlP2敏感性表征。这项拨款的续期将使我们能够从机制上深入了解不同的通道超家族如何依赖于PIP2。

DESCRIPTION (provided by applicant): The signaling phospholipid PIP2 has been recently appreciated for its critical role in regulating the activity of many ion channel and transporter proteins. The molecular details of how PTP2 controls the activity of such a diverse number of transmembrane proteins are unclear. Recent advances in the elucidation of the three-dimensional structure of a number of ion channel proteins makes it possible to study this problem at an atomic level of detail. Here we propose to identify the two end points of K channel gating by PIP2: the channel interaction sites with PTP2 and the channel gate that they control. First we will identify the channel gate that PIP2 controls in inwardly rectifying K (Kir) channels by using electrophysiology and mutagenesis coupled with a Substituted Cysteine Accessibility Method (SCAM) to probe state dependent modification during channel gating by PIP2. Secondly we will study the allosteric effects of Na gating in Kir3 and other Kir channels and the atomic details of Kir channel-PIP2 interactions. These studies will enable us to understand such functional consequences of these interactions, as the apparent channel affinity for PIP2 and the phosphoinositide stereospecific interactions with distinct channel sites. In close collaboration with computational biologists in our department, we have developed a combined electrophysiological and theoretical approach to analyze PIP2-sensitive channels: we first define their PIP2 interacting regions by computing molecular interaction fields of channels with phosphate probes; using a complementary computational approach we run Brownian Dynamics (BD) simulations using phosphoinositide probes to define the PIP2 interacting residues. We compare these two computational approaches and their agreement justifies pursuit of analysis at the next level, namely the refinement of the BD structures by Molecular Dynamics simulations to generate reliable models that can be tested experimentally. The validity of this combined experimental/theoretical approach has been developed and reinforced in Kir channels, where we have generated laborious experimental evidence of amino acid residues involved in interactions with PIP2. In the present competitive continuation of this work we propose to extend these studies to the family of voltage-gated K (Kv) channels, particularly because of the availability of a new Kv1.2 crystal structure. We provide preliminary data to show that different Kv subfamily members are PIP2-sensitive and propose a similar characterization of PlP2 sensitivity as with the Kir channels. Renewal of this grant award will allow us to gain mechanistic insights into how different channel superfamilies are dependent on PIP2.