Activation mechanism in HCN channels.

HCN 通道中的激活机制。

• 批准号: 10200855
• 负责人: Hans Peter Larsson
• 金额: $ 36.73万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200855
• 关键词: Antiepileptic Agents; Arrhythmia; Brain; C-terminal; Charge; Coupling; Cryoelectron Microscopy; Cyclic Nucleotides; Cysteine; Data; Dependence; Development; Drug Targeting; Epilepsy; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorometry; Free Energy; HCN1 gene; Heart; Human; Hydrogen Bonding; Ion Channel; Link; Measures; Membrane; Modeling; Molecular Conformation; Movement; Mutate; Mutation; Periodicity; Reagent; Structure; Testing; Transition Elements; crosslink; cyclic-nucleotide gated ion channels; fluorophore; molecular modeling; sensor; voltage; voltage clamp

Summary/Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain. Mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K+ (Kv) channels. Hyperpolarization-activated HCN channels and depolarization- activated Kv10.1 (EAG) channels have very similar tetrameric structures with six transmembrane segments (S1-S6) per subunit: S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD). In both Kv and HCN channels, S4 is the positively charged voltage sensor and the C-terminal part of S6 forms the gate. However, why Kv channels are activated by depolarization whereas HCN channels are activated by hyperpolarization is not clear. Using voltage clamp fluorometry, FRET, and cysteine accessibility and crosslinking, we will here measure the movement of S4 and the gate in HCN channels to determine the mechanism of activation of HCN channels by hyperpolarization. Our main hypothesis is that small differences in free energy between the closed and open states, due to different interactions between S4 and the pore in the different channels determines whether an ion channel opens by hyperpolarizations or depolarizations. Using recent structures of HCN and HCN-related channels as guide, we will mutate residues at the voltage sensor-pore domain interface and measure the effect of these mutations on voltage activation in HCN channels. In support of our main hypothesis, we show that mutations of only two residues located at the interface between this region of S4 and the PD reverse the voltage dependence of HCN channels so that the channels now open upon depolarization instead of hyperpolarization. We also hypothesize that the main S4 movement is not sufficient to open the gate, but that a second S4 movement is necessary for gate opening. We will measure these two different voltage sensor movements in HCN channels using membrane- impermeable cysteine reagents or fluorophores attached to S4, to determine what conformational change of S4 is necessary to open the gate in HCN channels. Using the recent structures of HCN and HCN-related channels, we will created molecular models of the different states of HCN channels to suggest a specific conformational change of S5 and S6 during gate opening. We will test the hypothesized gate movement using cysteine crosslinking between different channel domains. We will also measure conformational changes using unnatural fluorescent ANAP as the donor and transition metals as acceptors to conduct transition metal FRET between different channel domains. A better understanding of HCN channel gating will aid in development of better anti-arrhythmic and anti-epileptic drugs targeting HCN channels.

摘要/摘要 超极化激活的循环核苷酸门控（HCN）通道对于在节奏活动中至关重要 心脏和大脑。 HCN通道中的突变与心律不齐和癫痫有关。 HCN通道属于 给电压门控的K+（KV）通道的家族。超极化激活的HCN通道和去极化 - 活化的KV10.1（EAG）通道具有非常相似的四聚体结构，具有六个跨膜段 （S1-S6）每个亚基：S1-S4形成电压感应域（VSD）和S5-S6形成孔域（PD）。在 KV和HCN通道S4都是带正电压的电压传感器和S6形式的C末端部分 大门。但是，为什么通过去极化激活KV通道，而HCN通道则被激活 超极化尚不清楚。使用电压夹具荧光测定法，FRET和半胱氨酸可及性，并且 交联，我们将在这里测量S4和HCN通道中的门的运动，以确定 通过超极化激活HCN通道的机理。我们的主要假设是很小的差异 由于S4与孔之间的不同相互作用，在封闭状态和开放状态之间的自由能中 不同的通道决定了离子通道是通过超极化还是去极化打开的。 使用HCN和HCN相关通道的最新结构作为指南，我们将在电压下突变残基 传感器孔域界面并测量这些突变对HCN电压激活的影响 频道。为了支持我们的主要假设，我们表明仅两个位于 S4和PD之间的接口逆转了HCN通道的电压依赖性，以便 通道现在在去极化而不是超极化时打开。我们还假设主要S4 运动不足以打开大门，而是第二个S4移动对于门开口是必需的。 我们将使用膜 - 在HCN通道中测量这两个不同的电压传感器运动 附着在S4上的不可渗透的半胱氨酸试剂或荧光团，以确定什么构象变化 S4对于打开HCN通道的门是必要的。使用HCN和HCN相关的最新结构 通道，我们将创建HCN通道不同状态的分子模型，以建议特定 门打开期间S5和S6的构象变化。我们将使用 半胱氨酸在不同的通道域之间交联。我们还将使用 不自然的荧光ANAP作为供体和过渡金属作为传导金属fret的受体 在不同的通道域之间。对HCN渠道门控的更好理解将有助于开发 靶向HCN通道的更好的抗心律失常和抗癫痫药。