Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry
Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry
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DOI：
10.1016/s0896-6273(00)00117-3
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发表时间：
2000-11-01
期刊：
影响因子：
16.2
通讯作者：
Zagotta, WN
中科院分区：
文献类型：
--
作者：
Zheng, J;Zagotta, WN
特定部位的荧光记录在理解信号蛋白的构象变化方面显示出巨大的希望。已报道的离子通道的应用仅限于整个卵母细胞的胞外部位。我们现在能够使用同步的位点特异性荧光记录和从内向外斑片的膜片钳电流记录来直接监测环核苷酸门控通道的细胞内域的门控运动。当荧光团共价连接到环核苷酸结合域和孔之间的位置时，可以可靠地观察到荧光信号。阴离子猝灭剂碘化物在沟道闭合状态下具有较高的猝灭效率，而阳离子猝灭剂铊离子在沟道开态下具有较高的猝灭效率。猝灭的状态和电荷依赖性表明，在CNG通道激活过程中，带电荷的残基或偶极残基在荧光团附近移动。
Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patch-clamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.