The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.
The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.
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DOI：
10.1085/jgp.201611701
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发表时间：
2017-02
期刊：
影响因子：
--
通讯作者：
Robertson GA
中科院分区：
文献类型：
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作者：
Zhao Y;Goldschen-Ohm MP;Morais-Cabral JH;Chanda B;Robertson GA
hEAG 1是KCNH离子通道家族的成员，其特征在于C-末端区域与环核苷酸结合结构域（CNBhD）具有同源性。Zhao等人表明，占据CNBhD结合口袋的“内在配体”促进通道的激活和开放状态。钾离子通道的ether-à-go-go或KCNH家族中的通道的特征在于与环核苷酸结合同源结构域（CNBhD）具有同源性的保守C末端结构域。取代环状核苷酸，两个氨基酸残基Y 699和L701占据结合口袋，形成“内在配体”。然而，CNBhD在KCNH通道门控中的作用仍不清楚，并且缺乏对内在配体的详细表征。在这项研究中，我们表明，突变Y 699和L701丙氨酸，丝氨酸，天冬氨酸，或甘氨酸损害人类EAG 1通道功能。这些突变体减缓通道激活，并将电导-电压（G-V）关系转移到更多的去极化电位。突变影响激活和G-V关系逐步，表明门控机制是敏感的CNBhD的多种构象。在两个位点（GG）处用甘氨酸取代，其消除了与结合口袋相互作用的侧链，也降低了电压预脉冲沿着活化途径填充更多预活化状态的能力（即，Cole-Moore效应），就像在深度静止状态下稳定电压传感器一样。值得注意的是，整个CNBhD（577-708，ΔCNBhD）的缺失表型模仿GG突变体，表明GG是一种功能缺失突变，CNBhD需要内在配体才能发挥其功能作用。我们为野生型和ΔCNBhD突变体通道开发了一个动力学模型，该模型描述了我们对激活动力学、Cole-Moore位移和G-V关系的所有观察。这些发现支持CNBhD既促进电压传感器激活又稳定开孔的模型。内在配体对于这些功能效应是至关重要的。
hEAG1 is a member of the KCNH family of ion channels, which are characterized by C-terminal regions with homology to cyclic nucleotide–binding domains (CNBhDs). Zhao et al. show that an “intrinsic ligand” occupying the CNBhD binding pocket promotes the activated and open state of the channel. Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide–binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an “intrinsic ligand.” The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance–voltage (G–V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole–Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577–708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole–Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.