Molecular Mechanisms of Potassium Channel Permeation and Gating

摘要 钾(K+)通道是细胞兴奋性的主要决定因素，在生理学中起着至关重要的作用 流程。具体地说，大电导和钙激活的钾(BK)通道具有偶联的能力 细胞内钙离子到膜电位的变化，起着主要的生理作用，从血管 平滑肌张力的维持和昼夜节律的调节，到听力，调节神经元放电， 和神经递质的释放。BK通道功能障碍与许多病理生理机制有关 因此，了解钙门控可能会对治疗产生重大影响。总的目标是 这项授权是为了了解K+通道(打开、关闭和关闭)中钙门控的分子机制 失活)通过在模型BK通道上采用功能、结构和计算分析来实现 紧密的原核同源。与BK通道不同，在BK通道中，电压依赖门控干扰钙离子门控 因此，MthK没有电压传感器，因此无法确定特定构象的结构 一个单独研究钙依赖门控的完美系统。此外，电生理学实验 提示MthK和BK一样，缺乏选择性过滤器的失活。这很耐人寻味，因为BK和 MthK在过滤器中与其他失活的K+通道具有很高的序列和结构相似性。但是，在 在K+和双层厚度的特定条件下，MthK确实失活，增加了BK也 在这些条件下会失活。这可能会产生重大的生理后果，比如已知条件 这种控制活动将导致对BK通道在不同细胞类型和细胞中的作用的新的理解 地点。我们的第一个目标是确定钙激活的分子机制。我们建议确定 用单细胞凝胶电泳法研究了脂类纳米盘中重组通道中载脂蛋白和钙离子结合的MthK的结构。 粒子冷冻-EM。脂质成分将被调整，以产生开放状态。MD模拟将用于 优化脂膜的结构，模拟K+流量，并发现可能的激活途径。我们的 第二个目的是确定脂双层依赖的失活机制。我们将系统地 不同脂层厚度脂质体中MthK的活化与失活动力学研究 脂类长度。初步的停流功能数据显示，较薄的双层促进MthK失活。 利用单颗粒低温电子显微镜，我们将确定不同脂质组成的纳米盘中MthK的结构 并直接从功能分析中关联功能状态。MD模拟将完善这些结构，因为 以及观察膜厚度变化对构象和电导的影响。我们的第三个目标是 为了了解导致MthK失活的更细微的变化，我们建议使用X射线结晶学 结合MD模拟揭示了分子变化的序列 选择性过滤器，降低闸门、离子和水，通过施加促进功能失活的条件 化验。这些目标的实现将为K+通道中的钙门控提供一个全面的图景。

ABSTRACT Potassium (K+) channels are major determinants of cell excitability and play crucial roles in physiological processes. Specifically, large conductance and Ca2+-activated K+ (BK) channels, have the ability to couple intracellular Ca2+ to membrane potential variations, play major physiological roles ranging from vascular smooth muscle tone maintenance and regulation of circadian rhythms, to hearing, regulating neuronal firing, and neurotransmitter release. BK channel dysfunction has been associated with many pathophysiological conditions, so understanding Ca2+-gating can have major therapeutic consequences. The overall objective of this grant is to understand molecular mechanisms of Ca2+-gating in K+ channels (opening, closing, and inactivation) by employing functional, structural, and computational analysis on a model BK channel, MthK, a close prokaryotic homolog. Unlike BK channels, where voltage-dependent gating is interfering with Ca2+-gating thus preventing structural determination of specific conformations, MthK is devoid of voltage sensors and thus a perfect system for investigating Ca2+-dependent gating alone. In addition, electrophysiology experiments suggested that MthK, just like BK, lacks inactivation at the selectivity filter. This is intriguing, because BK and MthK share high sequence and structure similarity in the filter with other “inactivating” K+ channels. However, in specific conditions of K+ and bilayer thickness, MthK does inactivate, raising the possibility that BK also inactivates under these conditions. This can have major physiological consequences, as knowing conditions that control activity will lead to new understanding of BK channels’ role in different cell types and cellular locations. Our first aim is to determine the molecular mechanism of Ca2+-activation. We propose to determine the structures of apo and Ca2+ bound MthK, from channels reconstituted in lipid nanodiscs, using single- particle cryo-EM. Lipid composition will be adjusted to yield an open state. MD simulations will be employed to refine the structures in the lipid membrane, simulate K+ flux, and uncover possible activation pathways. Our second aim is to determine the lipid bilayer-dependent inactivation mechanism. We will systematically investigate MthK activation and inactivation kinetics in liposomes of varying lipid thickness made by varying lipid lengths. Preliminary stopped-flow functional data revealed that thinner bilayers promote MthK inactivation. Using single-particle cryo-EM, we will determine structures of MthK in nanodiscs of different lipid composition and associate functional states directly from functional assays. MD simulations will refine these structures, as well as observe how changing membrane thickness affects conformation and conduction. In our third aim, to understand the more subtle changes that lead to inactivation in MthK, we propose to use X-ray crystallography of pore-only MthK together with MD simulations to reveal the sequence of molecular changes involving selectivity filter, lower gate, ions and water, by imposing conditions that promote inactivation in functional assays. The accomplishment of these aims will provide a comprehensive picture of Ca2+-gating in K+ channels.