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Activation mechanism in HCN channels.

HCN 通道中的激活机制。

基本信息

批准号：
10609893
负责人：
Rene Barro-Soria
金额：
$ 36.73万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
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

项目摘要

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/摘要超极化激活的环核苷酸门控（HCN）通道是心脏节律性活动所必需的。心脏和大脑HCN通道的突变与心律失常和癫痫有关。HCN通道属于属于电压门控K+（Kv）通道家族。超极化激活的HCN通道和去极化- 激活的Kv10.1（EAG）通道具有非常相似的四聚体结构，具有六个跨膜片段（S1-S6）每个亚基：S1-S4形成电压敏感域（VSD），S5-S6形成孔域（PD）。在 Kv和HCN通道，S4是带正电荷的电压传感器，S6的C端子部分形成大门然而，为什么Kv通道被去极化激活，而HCN通道被去极化激活，超极化不清楚。使用电压钳荧光法、FRET和半胱氨酸可及性，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通道的机制。我们的主要假设是，由于S4和孔之间的不同相互作用，不同的通道确定离子通道是通过超极化还是去极化打开。使用HCN和HCN相关通道的最新结构作为指导，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中电压激活的影响渠道在支持我们的主要假设，我们表明，只有两个残基的突变位于 S4的该区域与PD之间的界面反转HCN通道的电压依赖性，使得通道现在在去极化而不是超极化时打开。我们还假设主要的S4 运动不足以打开闸门，但需要第二次S4运动才能打开闸门。我们将使用膜测量HCN通道中这两种不同的电压传感器运动- 不可渗透的半胱氨酸试剂或荧光团连接到S4，以确定什么样的构象变化， S4是打开HCN通道中的门所必需的。利用HCN和HCN相关的最新结构，通道，我们将创建HCN通道的不同状态的分子模型，以提出一个特定的 S5和S6在门打开期间的构象变化。我们将测试假设的门运动，不同通道结构域之间的半胱氨酸交联。我们还将测量构象变化，非天然荧光ANAP作为供体，过渡金属作为受体，进行过渡金属FRET 在不同的频道之间。更好地理解HCN通道门控将有助于开发更好的抗癫痫和抗癫痫药物靶向HCN通道。