Structural dynamics in cyclic nucleotide-modulated channels

摘要 环核苷酸调节通道在心脏和大脑的起搏活动中起重要作用， 神经系统中的嗅觉和视觉信号传导。这些渠道的功能缺陷导致 癫痫、心律不齐和色盲等疾病。这笔赠款的总体目标是 了解环核苷酸的结合如何门控（打开/关闭）通道以及其他因素，如 脂质和脯氨酸异构化调节该门控。我们将结合最先进的 技术：单粒子低温电子显微镜（cryo-EM）与原子力显微镜力谱 （AFM-FS）、天然质谱（MS）和功能测定如单通道电生理学和 掺入脂质体的通道的停流荧光测定。我们将聘请SthK，一个模型 原核环核苷酸调节通道，以及真核HCN 1和HCN 2用于选择子目标。我们 第一个目的是确定SthK中部分激动作用和配体选择性的分子机制。我们将 确定特定电压传感器SthK突变体的结构，这些突变体显示出增加的开放概率， 把班级平均值和单通道电生理学联系起来为了确定 配体选择性，我们将使用AFM-FS在单分子水平上测定cAMP和 cGMP单独或在全长通道的情况下与SthK环核苷酸结合结构域结合。这 将产生两个环核苷酸结合的能量学，并将分离孔对 约束力这一目标将阐明为什么cAMP结合不能完全打开SthK通道，以及为什么cGMP不能完全打开SthK通道。 一种拮抗剂，尽管其与结合口袋的结合方式与cAMP的结合方式相似。我们的第二个目标是 了解脂质如何调节通道活性。我们将系统地测试脂质对SthK活性的影响 使用停流荧光分析和单通道电生理学，其中通道在脂质体中 控制成分。我们将使用以下方法确定与通道（SthK和HCN 1）紧密结合的脂质： 天然MS，并确定它们如何通过扰动似乎 用功能测定来协调这些脂质-蛋白质相互作用。第三个目标是从功能上描述 在结构上，SthK以及潜在的HCN通道通过一种新发现的方式进行调节：脯氨酰 环核苷酸结合结构域中保守脯氨酸的异构化，这似乎是负责 用cAMP激活SthK的双相性这可能是非常有影响力的，因为脯氨酸异构化可能会导致 是另一种调节心脏和大脑起搏活动的方法。所有目标都是为了 阐明环核苷酸调节通道协同调节的分子机制， 配体、脂质和酶，其整合以产生生理学所需的通道激活水平， 牢房

ABSTRACT Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to understand how binding of cyclic nucleotides gates (opens/closes) the channels and how other factors such as lipids and proline isomerization modulate this gating. We will accomplish this by combining state-of-the-art techniques: single-particle cryo electron microscopy (cryo-EM) with atomic force microscopy force spectroscopy (AFM-FS), native mass spectrometry (MS), and functional assays like single-channel electrophysiology and stopped flow fluorescence assays of channels incorporated in liposomes. We will employ SthK, a model prokaryotic cyclic nucleotide-modulated channel, and also eukaryotic HCN1 and HCN2 for select sub-aims. Our first aim is to determine the molecular mechanisms for partial agonism and ligand selectivity in SthK. We will determine the structures of specific voltage-sensor SthK mutants that display increased open probability and correlate class averages with the single-channel electrophysiology. To determine the molecular mechanism for ligand selectivity we will use AFM-FS to determine at the single-molecule level the binding kinetics of cAMP and cGMP to either the SthK cyclic nucleotide binding domain alone or in the context of the full-length channel. This will yield the energetics of binding of both cyclic nucleotides and will isolate the contribution of the pore to the binding. This aim will shed light on why cAMP binding does not fully open the SthK channel and why cGMP is an antagonist, although its binding modality to the binding pocket is similar to that of cAMP. Our second aim is to understand how lipids modulate channel activity. We will systematically test the effect of lipids on SthK activity using stopped-flow fluorescence assays and single-channel electrophysiology where channels are in liposomes of controlled composition. We will determine the lipids tightly bound to the channels (both SthK and HCN1) using native MS and determine the mechanism of how they increase activity by perturbing the residues that appear to coordinate these lipid-protein interactions with functional assays. The third aim is to characterize functionally and structurally the regulation of SthK as well as potentially HCN channels by a newly discovered modality: prolyl isomerization of a conserved proline in the cyclic nucleotide binding domain, which appears to be responsible for SthK’s biphasic activation with cAMP. This can be highly impactful, as proline isomerization may turn out to be yet another means to regulate pacemaking activity in the heart and brain. All aims are geared towards unravelling the molecular mechanisms of cyclic nucleotide-modulated channels’ synergistic regulation by ligands, lipids and enzymes, which integrate to yield the channel activation levels required by the physiology of the cell.