How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels
How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels
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DOI：
10.1038/nchembio.747
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发表时间：
2012-02-01
影响因子：
14.8
通讯作者：
Benndorf, Klaus
中科院分区：
文献类型：
--
作者：
Kusch, Jana;Thon, Susanne;Benndorf, Klaus
超极化激活的环核苷酸调节（HCN）通道是在特化神经元和心肌细胞中产生电节律的四聚体膜蛋白。通道主要由电压激活，但也是受体，结合细胞内配体环AMP。通道激活的分子机制仍然是未知的。在这里，我们分析了复杂的激活机制的同源四聚体HCN 2通道共聚焦膜片钳荧光和动力学量化所有配体结合步骤和封闭开放异构化的中间状态。对于第二，第三和第四配体的结合亲和力，我们的研究结果表明显着的协同性，分别在序列阳性，阴性和阳性。这种复杂的相互作用的亚基导致优先稳定的状态与零，两个或四个配体，并建议二聚体组织的活化过程：在二聚体的协同性是积极的，而它是负的二聚体之间。
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.