Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.
Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.
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DOI：
10.1371/journal.pone.0185359
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Brelidze TI
中科院分区：
文献类型：
--
作者：
Hayoz S;Tiwari PB;Piszczek G;Üren A;Brelidze TI
超极化激活的环核苷酸调节（HCN）通道控制心脏和神经元的节律。HCN通道在其C-末端区域中含有环核苷酸结合结构域（CNBD），其通过C-接头连接至成孔跨膜区段。C-接头将由环核苷酸直接结合到HCN孔开口引起的构象变化偶联。最近，环二核苷酸被证明可以拮抗环核苷酸在HCN 4通道中的作用，但在HCN 2通道中不起作用。基于结构分析和突变研究，人们提出环状二核苷酸通过结合C-接头口袋（CLP）来影响HCN 4通道。在这里，我们首先表明，表面等离子体共振（SPR）可以用来准确地测量环核苷酸的C-接头/CNBD的HCN 2和HCN 4通道的结合亲和力。然后，我们使用SPR研究HCN通道中的环状二核苷酸结合。令我们惊讶的是，我们没有检测到环状二核苷酸与HCN 4通道的分离的单体C-接头/CNBD的结合。用等温量热法（ITC）进一步检查环状二核苷酸的结合，这表明环状二核苷酸与HCN 4通道的单体和四聚体C-接头/CNBD均不结合。两者合计，我们的研究结果表明，C-接头/CNBD与通道的其他部分的相互作用是必要的环二核苷酸结合在HCN 4通道。
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.
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影响因子：
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作者：
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DOI：
10.1073/pnas.1201997109
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