Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

中文翻译：

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HCN起搏器通道中相对亚基之间相互作用的功能和结构表征

超极化激活和环状核苷酸（HCN）调节通道是四聚阳离子通道。在四个亚基的每一个中，细胞内环状核苷酸结合结构域（CNBD）通过螺旋结构C接头与跨膜结构域偶联。高分辨率通道结构表明，C接头可实现与相对亚基的功能相关相互作用，这对于将CNBD中的构象变化偶联至通道孔可能至关重要。我们结合了诱变，膜片钳技术，共聚焦膜片钳荧光法和分子动力学模拟，以表明C接头的残基K464对于通过与相对亚基形成相互作用来稳定mHCN2通道的闭合状态至关重要。MD模拟显示，cAMP和K464E均诱导细胞内结构域相对于通道孔的旋转，从而削弱了未占据的CL-CNBD区域的自抑制作用。所采用的姿势与结构结果非常吻合。