Structural mechanisms for gating of bacterial cyclic nucleotide-gated ion channels

环核苷酸调节的离子通道是一种精密的分子机器 具有重要的生理功能。环核苷酸门控(CNG)通道 在光感受器和气味感受器中产生初级电反应 嗅觉受体。相关的超极化激活环核苷酸门控 (HCN)通道是心脏起搏活动的基础，心脏中的许多神经元 大脑。这些阳离子选择性通道是通过环状化合物的直接结合而打开的 核苷酸(cAMP和cGMP)连接到通道的胞内区。我们的目标是 揭示了CNG通道中这种变构的分子机制。我们的方法将 为研究细菌CNG通道作为真核通道的模型系统 因为它们为我们的生化方法提供了巨大的优势。我们会 利用四种不同方法的力量来确定结构， 构象异质性和这些通道的动力学：1)低温电子 显微镜(低温电子显微镜)，2)双电子-电子共振(DER)，3)微流控 与鹿结合的快速冷冻淬火(µRFQ)，以及4)基于Rosetta的分子 模特儿。该提案包括四名调查人员，他们都是这些领域的先驱 方法：研究方法。在同一离子通道上使用所有四种方法 条件是协同的，最终将导致全面的结构性和 这条通道变构的能量模型。归根结底是对分子的理解 这些通道不仅会告知心脏的生理学和病理生理学 和大脑，而且还有许多酶的变构控制的一般机制。

Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.