Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel.
Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel.
复制标题
超极化激活的 cAMP 门控通道配体进入路径附近致病突变的局部和全局解释。
DOI：
10.1016/j.str.2012.09.017
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Zhou,Lei
中科院分区：
文献类型：
--
作者：
Xu,Xinping;Marni,Farzana;Wu,Shengjun;Su,Zhuocheng;Musayev,Faik;Shrestha,Sabisha;Xie,Changan;Gao,Weihua;Liu,Qinglian;Zhou,Lei
超极化激活，cAMP门控（HCN）通道感知膜电位和细胞内cAMP水平。在人类HCN4通道的cAMP结合域（CNBD） S672R中发现的突变严重降低了心率，但其分子机制尚不清楚。我们对分离的CNBD进行的生化结合实验和功能通道上的膜片钳记录表明，S672R减少了cAMP的结合。突变体CNBD的晶体结构除了cAMP进入路径上的一个无序环外，没有显示出全局变化。为了在全蛋白水平上解决这种局部结构扰动，我们使用膜片钳荧光测定技术研究了cAMP与功能通道之间活性依赖的动态相互作用。S672R降低了cAMP在静息状态下与通道的结合，并显著提高了通道失活时的解结合率。这项对致病突变的研究说明了配体进出路径上的结构元件在稳定结合袋中的结合配体中所起的重要作用。
Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.