Structures of ion channel complexes

项目长期目标：更好地了解钾离子（K+）通道中与钙调素和KCNE亚基E(x)络合的正常门控的生物物理机制。我们描述了灵活的化学计量学，一种新的失活机制，Q1:E1通道复合物的变构门控，并在先前的授权期内采用了最先进的冷冻电镜技术。现在，我们将冷冻电镜与细胞生物物理方法相结合，如全细胞和单通道斑块记录、单卵母细胞荧光光谱、非天然氨基酸交联和分子动力学（MD）模拟，以解决四个短期目标：1)抑制剂的分子结合以快速激活Kv7.1通道（Q1）；了解HMR1556、UCL2077、异氟醚等抑制剂的作用机制，以及它们的直接或变构结合，将揭示Q1复合物是如何运作和被抑制的。能够获得Q1的高分辨率冷冻电镜结构，最终Q1:E(x)使我们能够测试不同抑制剂与各种通道化学计量络合的结构和生物物理性质。2) Q1+ E(x)通道化学计量固定复合物中激活门控的冷冻电镜和生物物理学：我们将开发现有的Q1冷冻电镜方法来解决第一个人类Q1:E(1)结构。由于Q1:E1通道形成了KCNQ1和KCNE(x)最重要的复合物（迄今为止尚未获得结构），这种结构将在调节通道复合物活性的化合物的设计和生物工程中具有广泛的科学用途。3)静息封闭态Kv7.1和Q1:E1通道结构的阐明：电压传感器（VS）和孔隙域是电压激活K+通道功能的两个重要结构元件，但目前还没有静息封闭K+通道与静息VS的结构表征。我们将使用E160R和/或E160R/R228E突变来抑制静息状态的通道，并获得封闭状态Q1:E(x)通道结构的冷冻电镜分辨率。4)通道动力学、药物结合/解除结合的马尔可夫和MD模拟：通道复合物的结构表征将指导进一步的低温电镜研究，并约束通道动力学的马尔可夫建模和MD模拟，使我们能够构建通道激活和抑制的动态模型。HQP：我们热情、严谨和多样化的学习环境将帮助HQP获得必要的技能，以实现独立和有价值的职业生涯。影响：低温电镜和生物物理研究Q1和Q1:E(x)抑制和门控将揭示这些生物看门人功能的分子事件，并提高对细胞电活动的理解。知识将通过出版物、邀请会议传播，这项工作预计将导致进一步的nserc合作机会，例如，IRAP、联盟，以产生进一步的科学、培训和经济机会。

Program Long-term Objective: To better understand biophysical mechanisms of normal gating in potassium Kv7.1 (KCNQ1 or Q1) ion (K+) channels, complexed with calmodulin and KCNE subunits, E(x). We described the flexible stoichiometry, a novel inactivation mechanism, the allosteric gating of the Q1:E1 channel complex, and adopted state-of-the art cryo-EM technology during the prior granting period. Now, we will combine cryo-EM with cellular biophysical methods such as whole-cell and single channel patch recording, single oocyte fluorescence spectroscopy, unnatural amino acid crosslinking, and molecular dynamics (MD) simulation, to address four short-term objectives: 1) Molecular binding of inhibitors to rapidly activating Kv7.1 channels (Q1): Understanding mechanisms of action of inhibitors such as HMR1556, UCL2077, isofluranes, and their direct or allosteric binding will reveal how Q1 complexes operate and are inhibited. Ability to obtain high resolution cryo-EM structures of Q1, and eventually Q1:E(x) allows us to test structural and biophysical properties of different inhibitors complexed with various channel stoichiometries. 2) Cryo-EM and biophysics of activation gating in stoichiometrically fixed complexes of Q1+ E(x) channels: We will develop our existing Q1 cryo-EM methodology to resolve the first human Q1:E(1) structure. As Q1:E1 channels form the most important complexes of KCNQ1 and KCNE(x) (no structures obtained to date), such a structure would have wide scientific utility in the design and bioengineering of compounds that modulate channel complex activity. 3) Elucidation of resting-closed state Kv7.1 and Q1:E1 channel structures: Two vital structural elements subserve the function of voltage-activated K+ channels: the voltage sensor (VS) and the pore domain, but currently there are no structural representations of resting closed K+ channels with resting VS. We will use the E160R and/or E160R/R228E mutations to restrain channels in their resting states and obtain cryo-EM resolution of closed-state Q1:E(x) channel structures. 4) Markov and MD simulations of channel dynamics, drug binding/unbinding: Structural representations of the channel complexes will guide further cryo-EM studies and constrain Markov modeling and MD simulations of channel dynamics, to allow us to construct dynamic models of channel activation and inhibition. HQP: Our welcoming, rigorous, and diverse learning environment will help HQP acquire necessary skills to achieve independent and rewarding professional careers. Impact: Cryo-EM and biophysical studies of Q1 and Q1:E(x) inhibition and gating will uncover molecular events underlying function of these biological gatekeepers, and improve understanding of cell electrical activity. Knowledge will be disseminated via publication, invited conference, and this work is anticipated to lead to further NSERC-partnered opportunities, e.g., IRAP, Alliance, to generate further scientific, training, and economic opportunities.