Laboratory of Physical Chemistry , ETH Zurich , 8093 Zurich , Switzerland.
School of Pharmacy , Chapman University , Irvine , California 92618 , United States.
J Am Chem Soc. 2019 May 8;141(18):7391-7398. doi: 10.1021/jacs.9b01092. Epub 2019 Apr 29.
In potassium (K) channels, permeation, selectivity, and gating at the selectivity filter are all governed by the thermodynamics and kinetics of the ion-protein interactions. Specific contacts between the carbonyl groups from the Thr-Val-Gly-Tyr-Gly signature filter sequence and the permeant ions generate four equidistant K binding sites, thereby defining the high ion selectivity and controlling the transport rate of K channels. Here, we used N-labeled ammonium (NH) as a proxy for K to study ion interaction with the selectivity filter of the prototypical full-length K channel KcsA by solution state NMR spectroscopy in order to obtain detailed insights into the physicochemical basis of K gating. We found that in the closed inactive state of KcsA (at pH 7) four K binding sites are occupied over a wide range of NH concentrations, while in intermediate closed-open conformations (at pH ∼6) the number and occupancy of K binding sites are reduced to two. However, in the presence of the scorpion toxin agitoxin II a total loss of NH binding is observed. NH titration studies allowed us to determine the dissociation constants of the four binding sites with values around 10 mM in the closed state of KcsA. Moreover, kinetic NMR experiments measured in the steady state equilibrium detected an off- and on-rate for NH of ca. 10 s and 10 s between KcsA-bound NH and the bulk. These findings reveal both the thermodynamics and kinetics of the ion binding sites and thus contribute to our understanding of the action of K channels.
在钾（K）通道中，渗透、选择性和门控在选择性过滤器处都受到离子-蛋白质相互作用的热力学和动力学控制。Thr-Val-Gly-Tyr-Gly 特征过滤器序列中的羰基与可渗透离子之间的特定接触产生了四个等距的 K 结合位点，从而定义了高离子选择性并控制 K 通道的转运速率。在这里，我们使用 N 标记的铵（NH）作为 K 的替代品，通过溶液态 NMR 光谱研究原型全长 K 通道 KcsA 中选择性过滤器与离子的相互作用，以深入了解 K 门控的物理化学基础。我们发现，在 KcsA 的关闭非活跃状态（在 pH 7 时），在 NH 浓度较宽的范围内，四个 K 结合位点被占据，而在中间关闭-开放构象（在 pH ∼6 时），K 结合位点的数量和占据减少到两个。然而，在存在蝎子毒素 agitoxin II 的情况下，观察到 NH 结合的完全丧失。NH 滴定研究使我们能够确定四个结合位点的离解常数，在 KcsA 的关闭状态下，其值约为 10 mM。此外，在稳态平衡下测量的动力学 NMR 实验检测到 NH 在 KcsA 结合的 NH 与主体之间的脱附和结合速率约为 10 s 和 10 s。这些发现揭示了离子结合位点的热力学和动力学特性，从而有助于我们理解 K 通道的作用。
