Localization of the pH gate in Kir1.1 channels.
Localization of the pH gate in Kir1.1 channels.
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DOI：
10.1529/biophysj.106.087700
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发表时间：
2006-10
影响因子：
3.4
通讯作者：
Yu‐Yang Zhang;H. Sackin;L. Palmer
中科院分区：
文献类型：
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作者：
Yu‐Yang Zhang;H. Sackin;L. Palmer
我们使用半胱氨酸修饰试剂来定位肾内向整流 K(+) 通道 Kir1.1a (ROMK1) 中的 pH 敏感门。细胞质侧甲硫磺酸盐 (MTS) 试剂可阻断爪蟾卵母细胞中表达的天然 Kir1.1 通道中的 K(+) 渗透。 N 末端、C 末端和跨膜结构域中的三个半胱氨酸的替换消除了对 MTS 试剂的这种敏感性（用由内而外的宏补丁测量）。在第二个跨膜结构域的 175-Kir1.1a 处重新引入一个半胱氨酸，允许 MTS 试剂 MTSEA、MTSET 和 MTSES 以及 Ag(+) 阻断开放通道。然而，低 pH 值导致的通道关闭可以保护其免受修饰。半胱氨酸也被引入到位置 G223，该位置被认为排列在通道的细胞质孔中。 MTSET 在打开和关闭状态下均阻止 G223C。相比之下，MTSEA 将 G223C 单通道电导从 40 pS 降低至 23 pS，但没有产生完全阻断。我们得出的结论是，细胞质酸化会引起通道蛋白的构象变化，从而阻止半胱氨酸修饰试剂（可能还有 K(+) 离子）从细胞质进入跨膜孔。这与 Kir1.1 pH 门在跨膜孔细胞质端附近的螺旋束交叉处的定位一致。
We used cysteine-modifying reagents to localize the pH-sensitive gate in the renal inward-rectifier K(+) channel Kir1.1a (ROMK1). Cytoplasmic-side methanethiosulfonate (MTS) reagents blocked K(+) permeation in native Kir1.1 channels, expressed in Xenopus oocytes. Replacement of three cysteines in the N-terminus, C-terminus, and transmembrane domains eliminated this sensitivity to MTS reagents, as measured with inside-out macropatches. Reintroduction of one cysteine at 175-Kir1.1a in the second transmembrane domain allowed blockade of the open channel by the MTS reagents MTSEA, MTSET, and MTSES and by Ag(+). However, closure of the channel by low pH protected it from modification. Cysteine was also introduced into position G223, which is thought to line the cytoplasmic pore of the channel. MTSET blocked G223C in both the open and closed state. In contrast, MTSEA reduced G223C single-channel conductance from 40 to 23 pS but did not produce complete block. We conclude that cytoplasmic acidification induces a conformational change in the channel protein that prevents access of cysteine-modifying reagents, and presumably also K(+) ions, to the transmembrane pore from the cytoplasm. This is consistent with localization of the Kir1.1 pH gate at the helix bundle crossing near the cytoplasmic end of the transmembrane pore.