Structural Determinants of PIP2 Regulation

描述（由申请人提供）：信号磷脂PIP 2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。PTP 2如何控制如此多样数量的跨膜蛋白的活性的分子细节尚不清楚。最近的进展，阐明了一些离子通道蛋白的三维结构，使人们有可能在原子水平上研究这个问题的细节。在这里，我们建议确定两个端点的K通道门控的PIP 2：与PTP 2的通道相互作用的网站和通道门，他们控制。首先，我们将确定通道门，PIP 2控制在内向整流K（Kir）通道，通过使用电生理学和诱变耦合取代半胱氨酸可及性方法（SCAM）探测状态依赖性修改通道门控期间由PIP 2。其次，我们将研究Na门控在Kir 3和其他Kir通道中的变构效应以及Kir通道-PIP 2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PIP 2的表观通道亲和力和磷酸肌醇与不同通道位点的立体特异性相互作用。在与计算生物学家在我们部门的密切合作，我们已经开发出一种结合电生理和理论的方法来分析PIP 2敏感的通道：我们首先定义他们的PIP 2相互作用区域通过计算分子相互作用领域的通道与磷酸探针;使用互补的计算方法，我们运行布朗动力学（BD）模拟使用磷酸肌醇探针来定义PIP 2相互作用的残基。我们比较了这两种计算方法，它们的一致性证明了在下一个层次上进行分析的合理性，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进行测试的可靠模型。这种结合的实验/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参与与PIP 2相互作用的氨基酸残基的费力的实验证据。在目前的竞争继续这项工作，我们建议将这些研究扩展到家庭的电压门控K（KV）通道，特别是因为一个新的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亚家族成员是PIP 2敏感的，并提出了类似的表征与Kir通道的PIP 2敏感性。更新该补助金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PIP 2。

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.