Activation mechanism in 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 心和脑。HCN通道的突变与心律失常和癫痫有关。HCN渠道属于 涉及电压门控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形成电压敏感结构域(VSD)，S5-S6形成孔域(PD)。在……里面 Kv和Hcn两个通道，S4是正电电压传感器，S6的C端部分形成 大门。然而，为什么Kv通道通过去极化激活，而hcn通道通过 超极化还不清楚。使用电压钳荧光法、FRET和半胱氨酸可及性 在这里，我们将测量S4和门在HCN通道中的移动，以确定 超极化激活HCN通道的机制。我们的主要假设是微小的差异 由于S4和孔In之间的不同相互作用，在闭合和开放状态之间的自由能 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。 以HCN及其相关通道的最新结构为指导，我们将在电压下突变残基 并测量这些突变对HCN中电压激活的影响 频道。为了支持我们的主要假设，我们证明只有两个残基的突变位于 S4的这个区域和PD之间的接口反转了HCN通道的电压依赖关系 频道现在在去极化时开放，而不是超极化。我们还假设主S4 移动不足以打开闸门，但需要第二次S4动作才能打开闸门。 我们将使用薄膜测量这两种不同的电压传感器在HCN通道中的运动- 不透性半胱氨酸试剂或结合到S4的荧光团，以确定什么构象变化 S4是打开HCN通道中的门所必需的。使用最新的hcn结构和hcn相关结构 通道，我们将创建不同状态的HCN通道的分子模型，以建议特定的 S5和S6在闸门开启过程中的构象变化。我们将使用以下命令测试假设的门移动 半胱氨酸在不同通道结构域之间的交联。我们还将使用以下工具测量构象变化 以非天然荧光ANAP为给体，过渡金属为受体进行过渡金属FRET 在不同的信道域之间。更好地了解HCN通道门控将有助于开发 更好的针对HCN通道的抗心律失常和抗癫痫药物。

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.