一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup> -</sup>通道和转运
Summary
Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.
Abstract
The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl– channels or transporters is described. The assay is based on the measure of the efflux rate of Cl– from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl– exchanger, as a model system. The efflux assay can be utilized to study any Cl– conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.
Introduction
在过去的二十年中，以过表达和纯化膜转运蛋白的能力显着增加：离子通道，初级和次级转运目前一般均从异源表达系统，以及天然来源纯化。新的方法来监测表达，改善和促进提取和提高这些蛋白质的稳定性正在不断开发1-5。这些技术的突破已经有助于触发的原子级的结构信息上的膜蛋白，这反过来，提高了我们自己的功能的结构基础的理解的爆炸。相比之下，我们的能力来探测纯化的蛋白质的功能性质没有增加以同样的速度，这样，在某些情况下，高分辨率的结构信息是伴随着定性功能数据，从而限制了我们的定量测试结构为基础的预测能力。因此，发展定量和可普及功能测定t是朝着膜蛋白功能的机械基础的澄清的关键步骤。
在这里，我们描述了可用于定量测定纯化和重组的Cl键的功能性质外排测定–通道和转运。该测定法的基本原则可以推广到各种运输系统，以及非离子转运蛋白。脂质体重组，纯化的Cl –的信道/转运在一个大的氯的存在下–梯度（ 图1A，B）。 Cl –的流出是通过加入一种离子载体的启动，以允许对离子通量，在我们的情况下，第K +离子载体缬氨霉素，其分流由氯建立的电压–梯度和设置初始膜电位至K的平衡电位+ 6,7。没有T他离子载体没有显著净Cl –的流出发生的，因为它防止了由一个跨膜电位的产生。 –流出，并且f 0，不含有活性蛋白的脂质体的分数τ，C1的时间常数：所述数据是由两个可测量参数（ 图1C）定量描述。从τ和f 0酉Cl –的传输速率，活性蛋白质的级分和活性复合体的分子质量可以衍生8。已知结构和功能的热交换器–该技术是利用脂蛋白重构与CLC-EC1，一个良好表征CLC型H + /氯这里示出。该测定是很容易推广到通道或转运与不同的离子选择性或其活性依赖于电压和/或配体的存在。另外，该测定可用于确定是否小分子直接影响重组蛋白，以定量这些化合物和如何膜组合物或脂质修饰试剂影响重构的通道和转运功能的影响。
Protocol
Representative Results
Discussion
我们已经描述了详细的协议来测量Cl –的运输由纯化的阴离子选择性通道或转运蛋白重构于脂质体介导的。所使用的例子是原核H + / Cl –的换热器CLC-EC1。然而，该方法可以容易地适合于研究通过配体12,13,15，11,12的电压，或者通过更换银运动不同的阴离子选择性15,16门控通道：与一种适合于所考虑的离子氯化银电极。电极选择性离子比其它氯– ，如H <su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Liposomicator, Avanti Polar Lipids Inc. | Avanti Polar Lipids Inc. | 610200 | |
| IEC Centra CL2 Benchtop | Thermo Scientific | ||
| Orion Research Model 701A Digital pH-mV meter | These can be found on Ebay. | ||
| Non-functional pH probe | Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned. | ||
| DI-710 Data Logger | DATAQ instruments | ||
| WinDAQ acquisition software | DATAQ instruments | ||
| Pierce Disposable Plastic Columns, Gravity-flow, 2ml | Pierce (Thermo Scientific) | 29922 | |
| KIMAX Culture Tubes, Disposable, Borosilicate Glass | Kimble Chase | 73500-13100 | |
| Extruder Set With Holder/Heating Block | Avanti Polar Lipids Inc. | 610000 | |
| Computer |
References
- Kawate, T., Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure. 14, 673-681 (2006).
- Drew, D., et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protocols. 3, 784-798 (2008).
- Hattori, M., Hibbs, R. E., Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure. 20, 1293-1299 (2012).
- Almo, S. C., Love, J. D. Better and faster: improvements and optimization for mammalian recombinant protein production. Curr Opin Struct Biol. 26, 39-43 (2014).
- Xiao, S., Shiloach, J., Betenbaugh, M. J. Engineering cells to improve protein expression. Curr Opin Struct Biol. 26, 32-38 (2014).
- Accardi, A., Miller, C. Secondary active transport mediated by a prokaryotic homologue of CLC Cl- channels. Nature. 427, 803-807 (2004).
- Nguitragool, W., Miller, C. Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions. J. Mol. Biol. 362, 682-690 (2006).
- Walden, M., et al. Uncoupling and turnover in a Cl-/H+ exchange transporter. J. Gen. Physiol. 129, 317-329 (2007).
- Accardi, A., Kolmakova-Partensky, L., Williams, C., Miller, C. Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels. J. Gen. Physiol. 123, 109-119 (2004).
- Basilio, D., Noack, K., Picollo, A., Accardi, A. Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter. Nat Struct Mol Biol. 21, 456-463 (2014).
- Lee, S. Y., Letts, J. A., MacKinnon, R. Functional reconstitution of purified human Hv1 H+ channels. Journal of Molecular Biology. 387, 1055-1060 (2009).
- Terashima, H., Picollo, A., Accardi, A. Purified TMEM16A is sufficient to form Ca2+ activated Cl- channels. Proc Natl Acad Sci U S A. 110, 19354-19359 (2013).
- Malvezzi, M., et al. Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nature Communications. 4, 2367 (2013).
- Picollo, A., Malvezzi, M., Houtman, J. C., Accardi, A. Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters. Nat Struct Mol Biol. 16, 1294-1301 (2009).
- Eckford, P. D., Li, C., Ramjeesingh, M., Bear, C. E. Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner. J Biol Chem. 287, 36639-36649 (2012).
- Stockbridge, R. B., et al. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci U S A. 109, 15289-15294 (2012).
- Menon, I., et al. Opsin is a phospholipid flippase. Curr Biol. 21, 149-153 (2011).
- Nimigean, C. M., Miller, C. Na+ block and permeation in a K+ channel of known structure. J Gen Physiol. 120, 323-335 (2002).
- Picollo, A., Xu, Y., Johner, N., Bernèche, S., Accardi, A. Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger. Nat Struct Mol Biol. 19, 525-531 (2012).
- Tsai, M. F., Fang, Y., Miller, C. Sided functions of an arginine-agmatine antiporter oriented in liposomes. Biochemistry. 51, 1577-1585 (2012).
- Heginbotham, L., Kolmakova-Partensky, L., Miller, C. Functional reconstitution of a prokaryotic K+ channel. J Gen Physiol. 111, 741-749 (1998).
- Lundbaek, J. A., Collingwood, S. A., Ingólfsson, H. I., Kapoor, R., Andersen, O. S. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes. J R Soc Interface. 7, 373-395 (2010).
- Lim, H. H., Stockbridge, R. B., Miller, C. Fluoride-dependent interruption of the transport cycle of a CLC Cl(-)/H(+) antiporter. Nat Chem Biol. 9, 712-715 (2013).
- Stockbridge, R. B., Robertson, J. L., Kolmakova-Partensky, L., Miller, C. A family of fluoride-specific ion channels with dual-topology architecture. Elife. 2, e01084 (2013).
- Nimigean, C., Shane, T., Miller, C. A cyclic nucleotide modulated prokaryotic K+ channel. J Gen Physiol. 124, 7 (2004).
- Brohawn, S. G., del Mármol, ., J, R., MacKinnon, Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K+ Ion Channel. Science. 335, 436-441 (2012).
- Jayaram, H., Robertson, J. L., Wu, F., Williams, C., Miller, C. Structure of a slow CLC Cl-/H+ antiporter from a cyanobacterium. Biochemistry. 50, 788-794 (2011).
