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

环核苷酸调节的离子通道是精致的分子机器， 是重要生理功能的基础环核苷酸门控（CNG）通道 在光感受器中产生对光的初级电反应， 嗅觉感受器相关的超极化激活的环核苷酸门控 (HCN)通道是心脏起搏器活动的基础，而心脏中的许多神经元 个脑袋这些阳离子选择性通道通过直接结合环 在一些实施方案中，将核苷酸（cAMP和cGMP）连接至通道的细胞内结构域。我们的目标是 揭示CNG通道变构的分子机制。我们的方法将 将细菌CNG通道作为真核通道的模型系统进行研究 因为它们为我们的生化方法提供了巨大的优势。我们将 利用四种不同的方法来确定结构， 构象异质性和这些通道的动力学：1）低温电子 显微镜（冷冻EM），2）双电子-电子共振（DEER），3）微流体 与DEER组合的快速冷冻淬灭（μ C），和4）基于Rosetta的分子 建模该提案包括四名调查人员，他们是每一项研究的先驱。 方法.使用所有四种方法在相同的离子通道下， 条件是协同作用，最终将导致全面的结构和 能量模型的变构这个渠道。最终， 这些通道不仅会告知心脏的生理学和病理生理学 和大脑，而且也是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.