The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms.
The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms.
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HCN 结构域是 HCN 通道细胞表面表达所必需的，并耦合电压和 cAMP 依赖性门控机制。
DOI：
10.1074/jbc.ra120.013281
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Brelidze,TinatinI
中科院分区：
文献类型：
--
作者：
Wang,Ze-Jun;Blanco,Ismary;Hayoz,Sebastien;Brelidze,TinatinI
超极化激活的环核苷酸门控(HCN)通道是心脏和脑中突触可塑性和节律性活动的主要调节因子。HCN通道的开放需要膜超极化，并被细胞内环核苷酸(CNMP)进一步促进。在HCN通道中，膜超极化是由跨膜电压传感器结构域(VSD)感知的，cNMP依赖的门控是由细胞内的环核苷酸结合结构域(CNBD)通过C-接头连接到成孔的S6跨膜片段而介导的。先前对HCN通道的功能分析表明，电压依赖和cNMP依赖的激活机制之间存在直接或变构耦合。然而，这种耦合的具体细节仍不清楚。HCN1通道的第一个冷冻-EM结构表明，一个新的结构元件，被称为HCN结构域(HCND)，在VSD和C-接头-CNBD之间形成了直接的结构联系。在这项研究中，我们探讨了HCND的功能意义。HCND的缺失阻止了HCN2通道的表面表达。根据HCN1的结构分析，我们确定了VSD S2上的Arg237和Gly239残基与HCND上的Ile135形成直接相互作用。破坏这些相互作用消除了HCN电流。我们还鉴定了C-接头-CNBD上的三个残基(Glu478、Gln482和His559)，它们与HCND上的Arg154和Ser158残基形成直接相互作用。破坏这些相互作用影响了电压依赖和cAMP依赖的HCN通道门控。这些发现表明，HCND对HCN通道的细胞表面表达是必需的，并在HCN通道门控的电压依赖和cAMP依赖机制之间提供了功能联系。
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker–CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237and Gly239residues on the S2 of the VSD that form direct interactions with Ile135on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker–CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154and Ser158on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.