Chaumont Severine, Khakh Baljit S
Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):12063-8. doi: 10.1073/pnas.0803008105. Epub 2008 Aug 8.
ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I(2) state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I(2) state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X(2) receptor I(2) state. However, P2X(2) receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins.
ATP 门控的 P2X 受体在受体激活后数秒内，随着通道进入 I(2) 状态，离子通透性增加，该状态对有机阳离子和染料分子具有通透性。这种重要行为背后的机制尚未完全理解。在一种模型中，I(2) 状态被认为是由于泛连接蛋白 1（Panx-1）通道的开放，而在另一种模型中，它被认为是 P2X 的固有特性。我们通过测量离子和染料通透性对这两种模型进行了测试，并使用膜片钳协同光谱方法来测量构象变化。我们的数据表明，Panx-1 通道对 P2X(2) 受体的 I(2) 状态没有可检测到的贡献。然而，P2X(2) 受体表现出通透性动力学，这与远离选择性过滤器本身的胞质结构域中的构象变化相关。最后，数据说明了一种新方法的实用性，即使用四半胱氨酸标签和双砷荧光团来测量膜蛋白中位点特异性的构象变化。
