Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6216-21. doi: 10.1073/pnas.0911270107. Epub 2010 Mar 8.
KcsA is a prokaryotic pH-dependent potassium (K) channel. Its activation, by a decrease in the intracellular pH, is coupled with its subsequent inactivation, but the underlying mechanisms remain elusive. Here, we have investigated the conformational changes and equilibrium of KcsA by using solution NMR spectroscopy. Controlling the temperature and pH of KcsA samples produced three distinct methyl-TROSY and NOESY spectra, corresponding to the resting, activated, and inactivated states. The pH-dependence of the signals from the extracellular side was affected by the mutation of H25 on the intracellular side, indicating the coupled conformational changes of the extracellular and intracellular gates. K(+) titration and NOE experiments revealed that the inactivated state was obtained by the replacement of K(+) with H(2)O, which may interfere with the K(+)-permeation. This structural basis of the activation-coupled inactivation is closely related to the C-type inactivation of other K channels.
KcsA 是一种依赖于 pH 的原核钾 (K) 通道。其激活是由细胞内 pH 值降低引起的，与随后的失活相偶联，但潜在的机制仍不清楚。在这里，我们通过使用溶液 NMR 光谱研究了 KcsA 的构象变化和平衡。通过控制 KcsA 样品的温度和 pH 值，产生了三个不同的甲基-TROSY 和 NOESY 光谱，分别对应于静息、激活和失活状态。来自细胞外侧的信号的 pH 依赖性受到细胞内侧 H25 突变的影响，表明细胞外和细胞内门的偶联构象变化。K(+)滴定和 NOE 实验表明，失活状态是通过用 H(2)O 取代 K(+) 获得的，这可能会干扰 K(+) 的渗透。这种激活偶联失活的结构基础与其他 K 通道的 C 型失活密切相关。
