Structural locus of the pH gate in the Kir1.1 inward rectifier channel
Structural locus of the pH gate in the Kir1.1 inward rectifier channel
复制标题
DOI：
10.1529/biophysj.104.051474
复制
发表时间：
2005-04-01
影响因子：
3.4
通讯作者：
Walters, DE
中科院分区：
文献类型：
--
作者：
Sackin, H;Nanazashvili, M;Walters, DE
原核生物内直肌的闭态晶体结构。ER，KirBac1.1，已经暗示细胞质一侧附近的四个内螺旋苯丙氨酸可能是通道门的一个位置。在目前的研究中，我们调查这种结构特征是否对应于肾内直肠的生理性pH门。呃，Kir1.1(ROMK，KCNJ1)。Kir1.1是哺乳动物肾脏集合管和Henle粗大上肢的内源性蛋白，在生理范围内受内部pH强烈调控。它有四个亮氨酸(L160-Kir1.1b)，与KirBac1.1的苯丙氨酸同源，这些亮氨酸可以在内部(M2)螺旋会聚附近作为立体门发挥作用。用较小的甘氨酸取代Kir1.1b的这些Leu-160残基可以消除pH门控；然而，用侧链介于亮氨酸和甘氨酸之间的丙氨酸取代并不能消除正常的pH门控。此外，通过将I163M-Kir1.1b突变添加到L160G突变构建的双突变也缺乏正常的pH门控，尽管I163M突变本身增强了通道的pH敏感性。除了大小，160-Kir1.1b的侧链疏水性对正常的pH门控也很重要。带有极性侧链的突变体(L160S、L160T)没有正常的门控，并且与L160G突变体一样对内部pH不敏感。因此，在160-Kir1.1b处的小或高极性侧链稳定通道的开放状态。基于KirBac1.1晶体结构的Kir1.1闭合状态的同源模型与我们的电生理数据一致，表明Kir1.1 pH门的关闭是由于四个亮氨酸在内跨膜螺旋的细胞质顶端汇聚而导致渗透途径的空间封闭。在打开状态下，K和它的水合壳一起穿过pH门。
The closed-state crystal structure of prokaryotic inward recti. er, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward recti. er, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.