This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Lipid Preparation
<ol>
	<li>Aliquot the desired amount lipids into a clear glass tube. Use E. coli polar lipid extract, but most lipid compositions can be used. If the lipids are in powder form, resuspend them in chloroform to a concentration of 20 mg/ml. Dry the lipids at RT under N2 gas until all solvent has evaporated.</li>
	<li>Resuspend the lipids in pentane and dry it again a steady stream of gas N2 to remove leftover traces of chloroform. Dry until all solvent has evaporated. Wh...

<ol>
	<li>Kawate, T., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-detection+size-exclusion+chromatography+for+precrystallization+screening+of+integral+membrane+proteins.">Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.</a> Structure. 14, 673-681 (2006).</li><li>Drew, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+optimization+scheme+for+the+overexpression+and+purification+of+eukaryotic+membrane+proteins+in+Saccharomyces+cerevisiae.">GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.</a> Nat Protocols. 3, 784-798 (2008).</li><li>Hattori, M., Hibbs, R. E., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-detection+size-exclusion+chromatography-based+thermostability+assay+for+membrane+protein+precrystallization+screening.">A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening.</a> Structure. 20, 1293-1299 (2012).</li><li>Almo, S. C., Love, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+and+faster:+improvements+and+optimization+for+mammalian+recombinant+protein+production.">Better and faster: improvements and optimization for mammalian recombinant protein production.</a> Curr Opin Struct Biol. 26, 39-43 (2014).</li><li>Xiao, S., Shiloach, J., Betenbaugh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cells+to+improve+protein+expression.">Engineering cells to improve protein expression.</a> Curr Opin Struct Biol. 26, 32-38 (2014).</li><li>Accardi, A., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+active+transport+mediated+by+a+prokaryotic+homologue+of+CLC+Cl-+channels.">Secondary active transport mediated by a prokaryotic homologue of CLC Cl- channels.</a> Nature. 427, 803-807 (2004).</li><li>Nguitragool, W., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+of+a+CLC+Cl-/H++exchange+transporter+by+polyatomic+anions.">Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions.</a> J. Mol. Biol. 362, 682-690 (2006).</li><li>Walden, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+and+turnover+in+a+Cl-/H++exchange+transporter.">Uncoupling and turnover in a Cl-/H+ exchange transporter.</a> J. Gen. Physiol. 129, 317-329 (2007).</li><li>Accardi, A., Kolmakova-Partensky, L., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+mediated+by+a+prokaryotic+homologue+of+CLC+Cl-+channels.">Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels.</a> J. Gen. Physiol. 123, 109-119 (2004).</li><li>Basilio, D., Noack, K., Picollo, A., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+required+for+H(+)/Cl(-)+exchange+mediated+by+a+CLC+transporter.">Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter.</a> Nat Struct Mol Biol. 21, 456-463 (2014).</li><li>Lee, S. Y., Letts, J. A., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+purified+human+Hv1+H++channels.">Functional reconstitution of purified human Hv1 H+ channels.</a> Journal of Molecular Biology. 387, 1055-1060 (2009).</li><li>Terashima, H., Picollo, A., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+TMEM16A+is+sufficient+to+form+Ca2++activated+Cl-+channels.">Purified TMEM16A is sufficient to form Ca2+ activated Cl- channels.</a> Proc Natl Acad Sci U S A. 110, 19354-19359 (2013).</li><li>Malvezzi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-dependent+phospholipid+scrambling+by+a+reconstituted+TMEM16+ion+channel.">Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.</a> Nature Communications. 4, 2367 (2013).</li><li>Picollo, A., Malvezzi, M., Houtman, J. C., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basis+of+substrate+binding+and+conservation+of+selectivity+in+the+CLC+family+of+channels+and+transporters.">Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters.</a> Nat Struct Mol Biol. 16, 1294-1301 (2009).</li><li>Eckford, P. D., Li, C., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J Biol Chem. 287, 36639-36649 (2012).</li><li>Stockbridge, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride+resistance+and+transport+by+riboswitch-controlled+CLC+antiporters.">Fluoride resistance and transport by riboswitch-controlled CLC antiporters.</a> Proc Natl Acad Sci U S A. 109, 15289-15294 (2012).</li><li>Menon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsin+is+a+phospholipid+flippase.">Opsin is a phospholipid flippase.</a> Curr Biol. 21, 149-153 (2011).</li><li>Nimigean, C. M., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na++block+and+permeation+in+a+K++channel+of+known+structure.">Na+ block and permeation in a K+ channel of known structure.</a> J Gen Physiol. 120, 323-335 (2002).</li><li>Picollo, A., Xu, Y., Johner, N., Bern&#232;che, S., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+substrate+binding+determines+the+stoichiometry+of+transport+of+a+prokaryotic+H(+)/Cl(-)+exchanger.">Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger.</a> Nat Struct Mol Biol. 19, 525-531 (2012).</li><li>Tsai, M. F., Fang, Y., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sided+functions+of+an+arginine-agmatine+antiporter+oriented+in+liposomes.">Sided functions of an arginine-agmatine antiporter oriented in liposomes.</a> Biochemistry. 51, 1577-1585 (2012).</li><li>Heginbotham, L., Kolmakova-Partensky, L., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+a+prokaryotic+K++channel.">Functional reconstitution of a prokaryotic K+ channel.</a> J Gen Physiol. 111, 741-749 (1998).</li><li>Lundbaek, J. A., Collingwood, S. A., Ing&#243;lfsson, H. I., Kapoor, R., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+regulation+of+membrane+protein+function:+gramicidin+channels+as+molecular+force+probes.">Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes.</a> J R Soc Interface. 7, 373-395 (2010).</li><li>Lim, H. H., Stockbridge, R. B., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride-dependent+interruption+of+the+transport+cycle+of+a+CLC+Cl(-)/H(+)+antiporter.">Fluoride-dependent interruption of the transport cycle of a CLC Cl(-)/H(+) antiporter.</a> Nat Chem Biol. 9, 712-715 (2013).</li><li>Stockbridge, R. B., Robertson, J. L., Kolmakova-Partensky, L., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+fluoride-specific+ion+channels+with+dual-topology+architecture.">A family of fluoride-specific ion channels with dual-topology architecture.</a> Elife. 2, e01084 (2013).</li><li>Nimigean, C., Shane, T., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyclic+nucleotide+modulated+prokaryotic+K++channel.">A cyclic nucleotide modulated prokaryotic K+ channel.</a> J Gen Physiol. 124, 7 (2004).</li><li>Brohawn, S. G., del M&#225;rmol, ., J, R., MacKinnon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Human+K2P+TRAAK,+a+Lipid-+and+Mechano-Sensitive+K++Ion+Channel.">Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K+ Ion Channel.</a> Science. 335, 436-441 (2012).</li><li>Jayaram, H., Robertson, J. L., Wu, F., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+slow+CLC+Cl-/H++antiporter+from+a+cyanobacterium.">Structure of a slow CLC Cl-/H+ antiporter from a cyanobacterium.</a> Biochemistry. 50, 788-794 (2011).</li></ol>

The authors declare no competing financial interests.

We have described a detailed protocol to measure Cl- transport mediated by purified anion-selective channels or transporters reconstituted in liposomes. The example used was the prokaryotic H+/Cl- exchanger CLC-ec1. However, the methodology can be readily adapted to study channels gated by ligands 12,13,15, voltage 11,12, or sporting different anionic selectivity 15,16 by replacing the Ag:AgCl electrode with one suitable for the ion under consideration. El...

In last two decades the ability to overexpress and purify membrane transport proteins has dramatically increased: ion channels, primary and secondary transporters are now routinely purified from heterologous expression systems as well as natural sources. New approaches to monitor expression, improve and facilitate the extraction and enhance stability of these proteins are constantly being developed 1-5. These technological breakthroughs have been instrumental in triggering the explosion of atomic-level structural information on membrane proteins which, in turn, enhanced our understanding of the structural bases of their function. In contrast, our ability to probe the functional properties of the purified proteins did not increase at the same rate, so that in some cases high resolution structural information is accompanied by qualitative functional data, thus limiting our ability to quantitatively test structure-based predictions. Hence, the development of quantitative and generalizable functional assays is a key step towards the elucidation of the mechanistic underpinnings of membrane protein function.
Here we describe an efflux assay that can be used to quantitatively determine key functional properties of purified and reconstituted Cl- channels and transporters. The principles underlying the assay can be generalized to a variety of transport systems, as well as to non ion-transporting proteins. Liposomes are reconstituted with purified Cl- channel/transporters in the presence of a large Cl- gradient (Figure 1A, B). Cl- efflux is initiated by the addition of an ionophore to allow for counter-ion flux, in our case the K+ ionophore valinomycin, which shunt the voltage established by the Cl- gradient and set the initial membrane potential to the equilibrium potential of K+6,7. Without the ionophore no significant net Cl- efflux occurs, as it is prevented by the generation of a transmembrane potential. The data is quantitatively described by two measurable parameters (Figure 1C): &#964;, the time constant of Cl- efflux, and f0, the fraction of liposomes not containing an active protein. From &#964; and f0 the unitary Cl- transport rate, the fraction of active proteins and the molecular mass of the active complex can be derived 8. The technique is illustrated here using proteoliposomes reconstituted with CLC-ec1, a well characterized CLC-type H+/Cl- exchanger of known structure and function. This assay is readily generalized to channels or transporters with different ionic selectivity or whose activity depends on the presence of voltage and/or ligands. Furthermore, this assay can be used to determine whether small molecules directly affect the reconstituted protein, to quantitate the effects of these compounds and how membrane composition or lipid-modifying reagents affect the function of the reconstituted channels and transporters.

<table border="0" cellpadding="0" cellspacing="0" width="995">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

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.

We describe a detailed and robust protocol to measure Cl- transport mediated by purified CLC-ec1, a prokaryotic CLC-type H+/Cl- exchanger, reconstituted in liposomes. A schematic representation of the experiment is shown in Figure 3. Proteoliposomes reconstituted with purified CLC-ec1 and containing high internal Cl- are immersed in a bath solution containing low Cl-. Under these conditions net Cl- efflux is prevented by the buildup of pos...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

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.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

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 ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

9.7K Görüntüleme.  Weill Cornell Medical College. 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.