Dynamic structural rearrangements of ion channels and receptors dependent on expression density.

我们研究的目的是阐明离子通道和受体功能随环境变化的机制，并了解它们的动态结构重排。在这笔拨款期间，我们进行了以下四项具体研究。[1]我们首次观察到ATP受体通道P2X2的孔道性质和配体敏感性可能是通过它们之间的相互作用而改变的，这可能取决于“膜上的开放通道密度”。我们还通过系统的突变研究发现，第二跨膜区的Ile328对表达密度依赖的变化至关重要。[2]利用杆状病毒-Sf9细胞表达系统纯化了P2X2重组蛋白，并用戊二醛锚定重组蛋白进行了化学计量分析。我们观察到，P2X2由三个亚基组成。我们还利用重组蛋白进行了单粒子结构分析，证实了P2X2是一个三聚体蛋白，呈倒金字塔状结构。[3]作为P2X2密度依赖性变化的一种可能机制，我们分析了磷脂依赖性。我们观察到，P2X2的功能不依赖于PIP2，而依赖于PIP3，并且扩张的孔道态与钝化态直接相关。[4]我们用FRET技术分析了代谢型谷氨酸受体的结构重排，观察到二聚体亚基的相对分配在谷氨酸的作用下发生了变化。我们还观察到mGluR1的偶联G蛋白的类型随着配体类型的不同而变化，表明mGluR1不是一个简单的开/关开关，而是一个多途径调节因子。

Our aim of research is to elucidate the mechanisms of the environment dependent changes of ion channels and receptor function and to know their dynamic structural rearrangements. During the period of this grant, we have conducted four specific researches in the following. [1]We, for the first time, observed that the pore properties and the ligand sensitivity of ATP receptor channel P2X2 change depending on "the open channel density on the membrane" probably by mutual interaction between them. We also uncovered by systematic mutagenesis study that Ile328 in the second transmembrane region is critical for the expression density dependent changes. [2]We purified recombinant protein of P2X2 by baculo virus - Sf9 cell expression system, and carried out stoichiometry analyses by anchoring the recombinant proteins with glutaraldehyde. We observed that P2X2 is composed of three subunits. We also performed single particle structure analysis using the recombinant proteins, and confirmed that P2X2 is a trimeric protein which shows an inverted pyramid like structure. [3]As a possible mechanism of the density dependent changes of P2X2, we analyzed phospholipids dependency. We observed that the P2X2 function depends not on PIP2 but on PIP3, and that the dilated pore state is directly linked to the desensitized state. [4]We analyzed the structural rearrangements of the metabotropic glutamate receptor by FRET technique, and observed that the relative allocation of the dimeric subunits changes upon glutamate application. We also observed that the type of coupling G proteins of mGluR1 changes depending on the type of ligand, showing that mGluR1 is not a simple on-/off- switch but a multiple path regulator.