Functional characterization of a ClC transporter by solid-supported membrane electrophysiology.
Functional characterization of a ClC transporter by solid-supported membrane electrophysiology.
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DOI：
10.1085/jgp.201210927
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发表时间：
2013-04
期刊：
影响因子：
--
通讯作者：
Dutzler R
中科院分区：
文献类型：
--
作者：
Garcia-Celma J;Szydelko A;Dutzler R
EcClC是ClC家族中的一个原核成员，是一种偶联的H+/Cl−交换器。由于已知的结构以及用不同的生物化学和生物物理技术研究其行为的可能性，该蛋白质已成为该家族的重要模型系统。虽然其功能的许多方面以前已被描述，但很难测量在不同环境条件下对同一样品的迁移。为了克服这个实验的局限性，我们研究了EcClC的固体支持膜电生理。大的运输相关的瞬态电流和一个简单的方式有关的运输速率的测量信号已经允许一个彻底的调查离子的选择性，抑制，和依赖于运输的离子浓度和pH值的变化。我们的研究结果证实，蛋白质运输较大的阴离子与约相似的速率，而较小的氟化物是不是一个基板。我们还发现，4，4 ′-二异硫氰基-2，2 ′-二苯乙烯二磺酸（DIDS），一种已知的其他阴离子转运蛋白的抑制剂，从细胞内侧不可逆地抑制EcClC。氯离子依赖性在毫摩尔浓度下表现出明显的饱和，这类似于真核ClC通道中的类似行为。我们的实验还使我们能够量化运输的pH依赖性。EcClC在低pH下显示出强活化，表观pKa为4.6。明显的pH依赖性是由一个保守的谷氨酸面临的细胞外的解决方案，以前被证明是一个受体转运质子的突变丢失，而它在很大程度上保留了在细胞内侧的等效残基的突变。我们的研究结果提供了一个定量的基础EcClC的运输行为，他们将作为一个参考，为未来的研究新的产电转运蛋白与仍然未表征的性质。
EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.
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影响因子：
2.9
作者：
Garcia-Celma, Juan J.;Ploch, Julian;Smirnova, Irina;Kaback, H. Ronald;Fendler, Klaus
通讯作者：
Fendler, Klaus
影响因子：
3.8
作者：
Accardi, Alessio;Walden, Michael;Nguitragool, Wang;Jayaram, Hariharan;Williams, Carole;Miller, Christopher
通讯作者：
Miller, Christopher
CLC Cl-/H+ 交换器中的细胞内质子转移突变体。
DOI：
10.1085/jgp.200810112
发表时间：
2009-02
期刊：
The Journal of general physiology
影响因子：
--
作者：
Lim HH;Miller C
通讯作者：
Miller C
DOI：
10.1085/jgp.102.4.631
发表时间：
1993-10
期刊：
The Journal of general physiology
影响因子：
--
作者：
Fendler K;Jaruschewski S;Hobbs A;Albers W;Froehlich JP
通讯作者：
Froehlich JP
影响因子：
64.8
作者：
Iyer, R;Iverson, TM;Miller, C
通讯作者：
Miller, C