Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA
Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA
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DOI：
10.1074/jbc.m608264200
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发表时间：
2007-05-18
影响因子：
4.8
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
--
作者：
Takeuchi, Koh;Takahashi, Hideo;Shimada, Ichio
离子通道的门控受到生理条件和细胞内/细胞外配体的严格调控。为了了解介导离子通道门控的潜在结构，我们利用溶液态核磁共振研究了K+通道KcsA在近生理条件下的ph依赖性门控。在不同pH值下测量的一系列(HN)-H-1-N-15-TROSY HSQC（横向弛豫优化光谱-异核单量子相干）光谱中，在pH 3.9和5.2之间检测到显著的化学位移变化，反映了与门控相关的构象重排。ph依赖的化学位移变化主要观察到细胞内螺旋束附近残基的共振，这被认为是形成K+通道的主要通道，以及内螺旋的细胞内延伸。Ala取代His-25消除了这种依赖于ph的构象重排，表明残基充当了通道的“ph传感器”。虽然KcsA的电生理开放概率小于10%，但在酸性和中性条件下，细胞内螺旋束的构象似乎有显著不同。这支持了最近提出的K+通道的“双门控”特性，其中选择性滤波器的激活耦合失活决定了通道的通道打开概率。实际上，还观察到来自Trp-67吲哚的信号的ph依赖性化学位移变化，该信号涉及与活化偶联失活相关的氢键网络。与爆发周期内的快速波动相比，胞内束的慢速动力学参数似乎更符合爆发持续时间的时间尺度，这表明存在另一种具有更快动力学特性的门控元件。
Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K+ channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (HN)-H-1-N-15-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K+ channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K+ channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.