Mechanism of proton gating of a urea channel
Mechanism of proton gating of a urea channel
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DOI：
10.1074/jbc.m312680200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Sachs, G
中科院分区：
文献类型：
--
作者：
Weeks, DL;Gushansky, G;Sachs, G
许多 pH 门控通道的大小和复杂性阻碍了特定结构模型的开发。肝螺杆菌的小型酸激活六膜段尿素通道 (HhUreI) 与胃病原体幽门螺杆菌的必需 UreI 同源，能够通过定点诱变识别质子门控的所有周质位点。暴露于外部酸度会增强表达 HhUreI 的爪蟾卵母细胞对 [C-14] 尿素的吸收，在 pH 6.8 时具有最大活性的一半 (pH(0.5))。单位点突变体中pH（0.5）的向下移动确定了影响质子门控的六个可质子化周质残基中的四个（第二跨膜片段TM2边界处的His-50、第一个周质环中的Glu-56、TM3边界处的Asp-59和TM6边界处的His-170）。 Asp-59 是唯一一个可质子化残基对于 pH 门控至关重要的位点。 PL2中Glu-110或Glu-114的突变不影响门控的pH(0.5)。嵌合体中 HhUreI 的整个周质结构域与唾液链球菌 UreI (SsUreI) 的膜结构域融合，保留了 SsUreI 的 pH 依赖性特性。因此，HhUreI 的质子门控可能取决于周质残基形成的氢键，进而产生跨膜结构域的构象变化。对 HhUreI 的进一步研究可能有助于了解其他生理上重要的 pH 响应通道。
The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [C-14]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.