Structural dynamics in cyclic nucleotide-modulated channels

摘要 环核苷酸调节的通道在心脏和大脑的起搏活动中起主要作用 神经系统中的嗅觉和视觉信号转导。这些渠道的功能缺陷导致 到癫痫、心律失常和色盲等疾病。这笔赠款的总体目标是 了解环核苷酸的结合如何开启/关闭通道，以及其他因素，如 脂类和脯氨酸异构化调节这种门控。我们将通过结合最先进的技术实现这一点 技术：单粒子低温电子显微镜(CRYO-EM)和原子力显微镜测力光谱 (AFM-FS)、自然质谱学(MS)和功能分析，如单通道电生理和 脂质体中通道的停流荧光分析。我们将雇佣SthK，一名模特 原核环核苷酸调控的通道，以及真核细胞的HCN1和HCN2亚靶标。我们的 第一个目的是确定SthK的部分激活性和配体选择性的分子机制。我们会 确定特定的电压传感器SthK突变体的结构，这些突变体表现出更高的开放概率和 将班级平均数与单通道电生理相关联。为了确定分子机制， 配体选择性我们将使用AFM-FS在单分子水平上确定cAMP和cAMP的结合动力学 CGMP单独与SthK环核苷酸结合域或在全长通道的上下文中结合。这 将产生两个环核苷酸结合的能量学，并将分离孔对 有约束力的。这一目标将阐明为什么cAMP结合不能完全开放SthK通道，以及为什么cGMP 拮抗剂，尽管其与结合口袋的结合方式类似于cAMP。我们的第二个目标是 了解脂质是如何调节通道活动的。我们将系统地测试脂质对SthK活性的影响 在脂质体中有通道的情况下使用停流荧光分析和单通道电生理 指受控制的成分。我们将使用以下方法确定与通道(SthK和HCN1)紧密结合的脂质 并确定它们如何通过干扰看起来像是 用功能分析来协调这些脂-蛋白相互作用。第三个目标是从功能上描述 在结构上，通过一种新发现的方式调节SthK和潜在的HCN通道：Prolyl 环核苷酸结合区中一个保守的脯氨酸的异构化，这似乎是原因 SthK与cAMP的双相激活。这可能是非常有影响的，因为脯氨酸异构化可能会导致 成为调节心脏和大脑起搏活动的另一种手段。所有目标都是面向 解开环核苷酸调控通道协同调控的分子机制 配体、脂类和酶，它们结合在一起，产生生理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.