Groupe d'étude des protéines membranaires (GÉPROM) and Department of Physics, Université de Montréal, Montreal, QC, Canada H3C 3J7.
Proc Natl Acad Sci U S A. 2013 May 14;110(20):8272-7. doi: 10.1073/pnas.1220398110. Epub 2013 Apr 29.
Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.
基于晶体学和分子动力学模拟的初始状态和最终状态的静态结构之间的线性插值，建立了电压门控钾通道（Kv）的原子级模型，因此缺乏动态结构信息。缺乏关于动力学和中间状态的信息，使得难以将电生理学获得的结构与动态功能数据联系起来。尽管电压钳荧光法填补了这一空白，但当门控的关键区域位于通道的胞质侧时，该方法仅限于可从细胞外进入的部位。在这里，我们通过使用荧光非天然氨基酸进行电压钳荧光法解决了这个问题。通过使用正交的 tRNA 合成酶对，将荧光非天然氨基酸掺入到 Shaker 电压门控钾通道中，该通道位于以前无法进入的关键区域。因此，我们确定了哪些部分独立作用，哪些部分协同作用，并发现孔的打开分两步进行。
