From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, and.
Departments of Structural Molecular Science and.
J Biol Chem. 2018 May 4;293(18):6969-6984. doi: 10.1074/jbc.RA118.001817. Epub 2018 Mar 15.
Canonical K channels are tetrameric and highly K-selective, whereas two-pore-domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K channels.
经典的 K 通道是四聚体且具有高度的 K 选择性，而双孔域钾通道（K2P）形成二聚体，但具有相似的孔结构。双孔域钾通道 TWIK1（KCNK1 或 K2P1）允许 Na 和其他单价离子通过，这主要归因于 P1 结构域中 Thr-118 的存在。然而，这种降低选择性的机制基础尚不清楚。我们使用离子交换诱导的差示红外光谱法分析了 WT TWIK1 和 T118I（高度 K 选择性）以及 L228F（P2 结构域中的取代）TWIK1 变体，并发现在存在 K 离子的情况下，WT 和两种变体在酰胺-I 带处都显示出 1680 cm 的峰。该峰对应于选择性过滤器中骨架羰基与 K 的相互作用，这一特征与经典 K 通道 KcsA 非常相似。计算分析表明，酰胺-I 带的相对高频很好地解释了与水分子形成氢键的破坏。此外，浓度依赖性光谱变化表明，WT 选择性过滤器的 K 亲和力远低于变体。此外，只有变体在从 K 到 Rb 或 Cs 条件的变化下显示出酰胺-I 带的更高频率位移。高速原子力显微镜揭示 TWIK1 的表面形貌在 K 和 Na 溶液中基本不变。我们的结果揭示了 TWIK1 选择性过滤器的局部构象变化，并表明酰胺-I 带可能是评估其他 K 通道特性的有用“分子指纹”。
