ClC-32Cl-/1H+交换剂调节内体pH和Cl-浓度。我们研究了ClC-3介导的离子传输(稳态传输电流,ISS),门控电荷(Q)和细胞质碱化。ClC-3转运在功能上是单向的。ClC-5和ClC-3显示出无法区分的交换比,但ClC-3循环效率较低,正如大型Q/ISS所反映的那样。M531A突变预测会增加水线稳定性,细胞质质子供应会提高效率。外谷氨酸门(Gluext;E224)的质子化(pH5.0)降低了Q,抑制运输,削弱耦合。中心酪氨酸阴离子门(Y572S)的去除大大增加了非偶联阴离子电流。酪氨酸-OH去除(Y572F)改变阴离子选择性并损害偶联。E224和Y572充当阴离子屏障,并有助于门控。Y572侧链和-OH调节Q运动动力学和电压依赖性。E224和Y572相互作用以创建在循环期间保持耦合的“封闭”内栅极构象。
我们利用质膜定位的ClC-3来研究稳态传输电流(ISS)之间的关系,门控电荷(Q)运动,和细胞质碱化率。ClC-3表现出比ClC-5更低的传输效率,这由更大的Q/ISS比反映出来,但无法区分Cl-/H+耦合比。外部SCN-降低了80-90%的H转运率和未偶联的阴离子/H交换。去除外部门控谷氨酸(“Gluext”)(E224A突变)降低了Q并消除了H转运。我们假设甲硫氨酸531(M531)阻碍了“水丝”H从细胞质转移到E224。因此,M531A突变使Q/ISS比降低50%,并增强H+转运。外部质子(pH5.0)抑制ISS并显着降低Q,同时正改变Q-电压(V)关系。pH5.0时Cl-/H+偶联比显著增加,与采用向外/开放位置的外部质子化Gluext一致。内部“负离子门”移除(Y572S)显著增加了ISS和受损的耦合,不减慢H+的传输速率。两个栅极(Y572S/E224A)的损耗导致大的“开孔”电导。Y572F(仅去除酚氢氧化物)和Y572S同样缩短了Q持续时间,导致在所有电压下更快的Q动力学。这些数据揭示了Q和离子传输之间的复杂关系。Q/ISS必须与耦合比一起评估,以正确解释效率。偶联和转运速率受阴离子的影响,内部质子供应和外部质子。Y572调节H+偶联以及阴离子选择性,并直接与E224交互。内部质子破坏这种“闭门”构象可能代表ClC-3运输循环中的关键步骤。
The ClC-3 2Cl- /1H+ exchanger modulates endosome pH and Cl- concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, ISS ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less \"efficient\", as reflected by a large Q/ISS . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Gluext ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a \"closed\" inner gate conformation that maintains coupling during cycling.
We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (ISS ), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/ISS ratio, but an indistinguishable Cl- /H+ coupling ratio. External SCN- reduced H+ transport rate and uncoupled anion/H+ exchange by 80-90%. Removal of the external gating glutamate (\"Gluext \") (E224A mutation) reduced Q and abolished H+ transport. We hypothesized that Methionine 531 (M531) impedes \"water wire\" H+ transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/ISS ratio by 50% and enhanced H+ transport. External protons (pH 5.0) inhibited ISS and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl- /H+ coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Gluext adopting an outward/open position. Internal \"anion gate\" removal (Y572S) dramatically increased ISS and impaired coupling, without slowing H+ transport rate. Loss of both gates (Y572S/E224A) resulted in a large \"open pore\" conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/ISS must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H+ coupling as well as anion selectivity, and interacts directly with E224. Disruption of this \"closed gate\" conformation by internal protons may represent a critical step in the ClC-3 transport cycle.