Gating the glutamate gate of CLC-2 chloride channel by pore occupancy
Gating the glutamate gate of CLC-2 chloride channel by pore occupancy
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DOI：
10.1085/jgp.201511424
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发表时间：
2016-01-01
影响因子：
3.8
通讯作者：
Arreola, Jorge
中科院分区：
文献类型：
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作者：
De Jesus-Perez, Jose J.;Castro-Chong, Alejandra;Arreola, Jorge
ClC-2通道是响应超极化而开放的二聚体双管氯离子通道。超极化激活了独立调节每个亚单位中孔的渗透性的原孔门和影响通过两个孔的渗透率的公共门。ClC-2通道缺乏经典的跨膜电压敏感结构域；相反，它们的原生孔门(位于孔内，每个由谷氨酸残基的侧链形成)在细胞内阴离子的排斥或细胞外H+的质子化作用下打开。在这里，我们证明了ClC-2的电压依赖的门控：(A)当胞液侧存在意向阴离子(Cl-、Br-、SCN-和I-)时；(B)发生在胞液侧的不良阴离子氟化物、谷氨酸、葡萄糖酸和甲烷磺酸；(C)取决于有意和意向不良的阴离子占据孔道；(D)强烈地促进多离子占据；(E)在可能的质子化条件下(pH(E)=5.5或6.5)在用醋酸盐(意指阴离子)透析的细胞中不存在；和(F)在细胞内pH 7.3和4.2时相同；以及(G)在不太可能的质子化条件下(Phe=10)暴露于增加[Cl-](I)的全细胞和由内向外的斑块中观察到。因此，根据我们的结果，我们认为超极化主要是通过驱动细胞内的阴离子进入通道孔来激活ClC-2，而细胞外H+的质子化在移位谷氨酸门的过程中起着次要的作用。
CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H+. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl-, Br-, SCN-, and I-) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pH(e) = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl-](i) under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H+ plays a minor role in dislodging the glutamate gate.