The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H+ Killing Selection
<ol>
	<li>Cell lines
	<ol>
		<li>Stably transfect NHE-deficient cells (e.g., the CCL39-derived PS120 cell line16) using any combination of mammalian expression vector and transfection method that will produce efficient transfection yields and selection in a fibroblast cell line. 
		NOTE: For many years Calcium Phosphate precipitation19 was used with good transfection yields. This has been replaced more recently by commercial reagents such as Lipofectamine2000, the transfections being performed following the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Solutions
	<ol>
		<li>Prepare three sterile solutions described as below. Sterilize these solutions by filtration (0.22 &#181;m).
		<ol>
			<li>Prepare a NH4+ Loading solution (pH 7.4) comprised of 50 mM NH4Cl, 70 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM MOPS at pH 7.4.</li>
			<li>Prepare a rinse Solution (pH 7.0) comprised of 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.0.</li>
			<li>Prepare a Recovery solution (pH 7.4) comprised of 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Before starting the selection, obtain a cell culture incubator that can be used at 37 &#176;C with no additional CO2 from external tank (termed CO2-&#8220;free&#8221; incubator), as 5% CO2 will result in buffering that may impair the acidification. Amplify the cell line expressing the NHE of interest up to about 2 x 108 cells (typically about 20 confluent 100 mm plates).</li>
	<li>H+ killing procedure:
	<ol>
		<li>Incubate the cells expressing the intracellular NHE of interest for 1 hour in the Loading Solution, at 37 &#176;C in the CO2-free incubator.</li>
		<li>Aspirate the loading solution and then rinse them twice in the above described-rinse solution. Perform this step efficiently to eliminate the residual extracellular NH4Cl that would impair the creation of a steep NH4+ gradient that will generate the acidification.</li>
		<li>Eliminate the rinse medium by aspiration and incubate the cells for one hour in recovery solution again at 37 &#176;C in the CO2 free incubator.</li>
		<li>Replace the recovery medium with regular culture medium (typically DMEM with 7.5% FCS) and grow the cells in standard culture conditions (37 &#176;C in a humidified atmosphere of 5% CO2 and 95% air).</li>
	</ol>
	</li>
	<li>Repeat this cycle of selection twice a week until stable clones emerge. 
	NOTE: Take special care concerning the cell culture and selection conditions. Months of work can be ruined by any contamination that is more prone to occur after a long period of culture and repeated cell manipulations.
	<ol>
		<li>Here, choose whether to amplify the clones and characterize them individually as further described in sections 2-3, or pool them together to generate a cellular population before characterization.</li>
	</ol>
	</li>
	<li>Apply occasional selection (about once every two weeks) to retain the acidification-resistant cells by counter-selecting those that may have reverted to the parental acid-sensitive phenotype (Figure 1B).</li>
</ol>
2. Intracellular pH Measurements
2.1) Fluorescence pH Imaging
<ol>
	<li>Use the ratiometric pH-sensitive fluorescent dye BCECF/AM, which is pH sensitive when excited at 490 nm and possesses a 445 nm isosbestic point, following manufacturer&#8217;s protocols.
	<ol>
		<li>Alternatively, use other pH&#8211;sensitive probes, following the manufacturer&#8217;s protocols.</li>
	</ol>
	</li>
	<li>Use an imaging set consisting of an inverted microscope coupled to a high sensitivity video camera. Equip this set with 450 nm and 490 nm narrow band interference filters paired with appropriate quartz neutral-density filters for excitation.
	<ol>
		<li>If possible, use the appropriate set of filters and illumination conditions to setup comparable fluorescence values for &#955;1 and &#955;2 so that the ratio approaches 1. Also avoid basal fluorescence values that are set either saturating or too low, both for &#955;1 and/or &#955;2. This may yield important artifacts as then the ratio may not been detectable although intracellular pH is changing. 
		NOTE: This system must enable rapid perfusion, time-lapse excitation at the two above-mentioned wavelengths and acquisition at the proper emission wavelength (535 nm for BCECF). Equip the system with software enabling automatic data acquisition and storage.</li>
	</ol>
	</li>
</ol>
2.2) Measurement of NHE Forward Activity (Extrusion of Protons from the Cytosol)
<ol>
	<li>Acidify cells by a 1 hr incubation in the NH4+ Loading solution (see step 1.4) in the absence of CO2.</li>
	<li>Add BCECF-AM (5 &#181;M final concentration) to the solution for the last five minutes and then rinse it with NH4+ loading solution to eliminate the extracellular probe.</li>
	<li>Mount the cells on the microscope and take several images to record a stable baseline. Perfuse the rinse solution (see above). This results in a drop of fluorescence corresponding to the acidification.</li>
	<li>After pH stabilization, perfuse any of the solutions given below to measure pH recovery rates mediated by Li+/H+, Na+/H+ or K+/H+ exchange. Each solution contains a potential coupling cation to be tested for H+ exchange. If one of those is transported, this will result in a fluorescence increase at 495 nm that will correspond to a raise in intracellular pH: 
	120 mM LiCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 
	120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 
	125 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4</li>
</ol>
2.3) Measurement of NHE Reverse Activity
NOTE: The principle here is to invert the transmembrane ionic gradients.
<ol>
	<li>Prior to measurement, load the cells with the intracellular cation of interest (see below).</li>
	<li>Incubate them for 5 min with BCECF/AM (see step 2.2.2); rinse them, mount them on the video microscope.</li>
	<li>Perfuse the loading solution and take several images to record a stable baseline.</li>
	<li>After the baseline stabilization, image the pH variations when cells are perfused with an extracellular acidic medium containing 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM MES at pH 6.5.</li>
	<li>For LiCl loading, incubate cells for 2 hr in a solution composed of 120 mM LiCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4. Intracellular lithium measured by atomic absorption spectroscopy yields a value of about 60 mM.</li>
	<li>For Na+ loading, incubate cells for two hours in a solution composed of 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 in the presence of 1 mM ouabain to block the Na/K ATPase. In these conditions, intracellular Na+ concentration is in the range of 40 mM. 
	NOTE: K+ loading is not necessary as cytosolic K+ concentration is in the range of 140 mM and an outwardly-directed K+ gradient is therefore easy to achieve.</li>
</ol>
2.4) Data Treatment and Calibration:
NOTE: This step applies both to the forward and reverse measurements.
<ol>
	<li>At the end of each experiment, perfuse cells with a solution of 140 mM KCl, 20 mM HEPES and 5 &#181;M nigericin adjusted to pH values between 6.5 and 7.4.</li>
	<li>Collect data as fluorescence measurements from the images (grey levels representing intensity levels) and export under text format and treat as explained below using a spreadsheet program.</li>
	<li>Calculate intracellular pH values for each individual cell or region of interest using the following equation: 
	pH = pKa + (Log (R-Rmin)/(Rmax-R))x Fmin(&#955;2)/Fmax(&#955;2)</li>
	<li>Use the Fmin(&#955;2)/Fmax(&#955;2) ratio if there is probe bleaching or leakage, resulting in a decrease in the 450 nm fluorescence throughout the experiments. This is however very rarely observed in the conditions used in the described experiments.</li>
	<li>As the calibration data are present at the end of each pH measurement, check whether the corresponding calculated values differ from the pH values used for calibration. If it is the case, then adjust all experimentally-determined pH values to the calibration using the following procedure:
	<ol>
		<li>Calculate the following quotient (Q, the difference between the measured values/Difference between the calibration values). Multiply the whole dataset by Q. Make the final adjustment by addition or subtraction so that the calculated values will equal the corresponding calibration values.</li>
	</ol>
	</li>
</ol>
3. Measurements of Initial Rates of NHE7 by Fast Li+ Uptake
<ol>
	<li>Seed cells on multi well plates (6 to 24-well) and acidify them using either the NH4+ loading technique described above or alternately the nigericin/bovine serum albumin (BSA) acidification described in20.</li>
	<li>Rinse plates twice rapidly using the above-described rinse solution and then incubate the cells in uptake solutions containing 1-10 mM lithium (slightly below Km values) and the desired concentrations of the cations and/or inhibitors of interest for the desired amount of time.</li>
	<li>Maintain short and consistent uptake durations (typically one minute or below) to ensure that transport operates in initial rates conditions. Also make sure that all the solutions used for uptake are isotonic.</li>
	<li>At the end of the uptake time, carefully eliminate the uptake medium and wash the cells four times with ice-cold phosphate buffered saline (PBS). Perform these washes as fast as possible (less than 10 sec for the four of them is ideal) to prevent lithium efflux.</li>
	<li>Lyse the cells in 25% Nitric acid. Apply 250 &#181;l per well of 25% Nitric acid and let it sit for at least 1 h, then scrap each well with the end of the pipette tip and transfer the whole well suspension in a new microcentrifuge tube.</li>
	<li>Transfer the lysate in 1.5 ml microcentrifuge tubes, centrifuge them for 5 minutes at 15,000 x g (room temperature) to remove the cellular debris.</li>
	<li>Measure the lithium content of the supernatants by atomic absorption spectroscopy21. 
	NOTE: Different companies provide atomic absorption spectrometers, which have to be equipped with a Lithium hollow cathode lamp. Follow the manufacturer&#8217;s instructions as these machines are very precise but also quite delicate.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes how to select cells expressing intracellular Na+/H+ exchangers at the plasma membrane without altering their primary sequence by site-directed mutagenesis. These exchangers can now be characterized.
This method is based on the cellular toxicity of intracellular protons to select cell lines that will express vesicular Na+/H+ exchangers at the plasma membrane. It might be possible, in principle, to use other cations that may be ...

Most intracellular compartments display an acidic luminal pH, which is a key parameter for maturation, trafficking, recycling of proteins or hormones and neurotransmitters loading. It has been shown that the pH gradient between cytosol and vesicular content is generated by vacuolar H+ ATPases1, coupled to vesicular ClC chloride transporters2. Both in knock-out (KO) mice and human patients, the importance of these transporters has been highlighted by the heavy phenotypes caused by mutations in their genes3-6.
The members of the Sodium-Hydrogen exchangers SLC9A family, also termed NHEs for Na+/H+ exchangers, have been shown to be key effectors in intracellular pH and cell volume regulation, as well as in vectorial transport of acid-base equivalents across epithelia. Besides the plasma membrane NHEs, three highly conserved Na+/H+ exchangers, NHE 6, 7 and 9 are expressed in trans-Golgi network and in early endosomes7. Mutations in their genes have been linked with Angelman-like or Christianson Syndromes8-9, family-based autism10 and Attention Deficit Hyperactivity Disorder11-12. These exchangers have also been involved in neurodegenerative problems such as Alzheimer disease susceptibility13 and X-linked mental retardation contiguous genes syndromes14. Taken together, these studies highlight the importance of these intracellular NHEs in brain development and/or function.
The intracellular localization of these exchangers prevents accurate measurements of their ion selectivity, transport direction, kinetic parameters, and regulation. As is the case for all transporters expressed in intracellular compartments, it is extremely difficult to assess their biochemical activities and hence to fully understand their physiological roles and the mechanisms underlying their pathological implications. Based on the high cytosolic K+ concentration, the most-commonly accepted hypothesis was that they were working as K+ coupled proton efflux transporters. The existence of such a proton leak had been hypothesized, as it may counterbalance proton pumping by the V-ATPases in order to maintain a steady state vesicular pH. The aim of this visual article is (i) to demonstrate a method that allows the genetic selection of cell lines that express such vesicular transporters at their plasma membrane, and (ii) to show two independent approaches to measure the functions of these transporters.
Three decades ago, Pouyss&#233;gur and Franchi have pioneered a genetic approach that enabled the molecular cloning and characterization of the members of the NHE family15. This was based on the toxicity of intracellular protons as a screening method. The first step was to obtain cell lines deficient in any Na+/H+ exchange expressed at the plasma membrane, using the reversibility of this transporter. Fibroblasts (CCL39 cell line) were preloaded with Na+ or Li+ and then placed in an acidic extracellular medium (pH 6.5) for 2 hr. This led to the death of cells expressing a functional Na+/H+ exchange and to the selection of antiporter-deficient cells (PS120 cell line)16. When cultivated in bicarbonate-free medium, these cells are very sensitive to acute intracellular acidification. Consequently, the expression of any functional proton efflux mechanism at the plasma membrane will be positively selected (see17) if such cells are submitted to acute intracellular acidifications. Such acidification techniques can be used to isolate cell lines with trafficking defects enabling the forced expression of WT intracellular NHEs at the plasma membrane.
As eukaryotic Na+/H+ exchangers are electroneutral, they are not measurable by the electrophysiological approaches that have been used with great success to measure channels. This manuscript therefore demonstrates how to measure the activity of this exchanger by intracellular pH measurements and rapid kinetics of lithium uptake. As the underlying concepts are the same, it is interesting to notice that many of the processes developed for the selection section are also used directly for functional measurements.
Interestingly, we have observed that the trafficking defect present in the cell lines selected using the approach described in this manuscript leads to a greater expression of other vesicular proteins at the plasma membrane such as the vesicular potassium channel TWIK118. This points out toward the selection of a general retention defect mechanism for vesicular transmembrane proteins. Hence this selection procedure and the cells that it generates may constitute a promising tool for the scientific community working on the membrane proteins of intracellular compartments. As well the measurement techniques presented here might be applicable for studying other non-electrogenic transporters.

<table><tbody><tr><td>Standard cell culture equipement</td><td></td><td></td><td>Used for H+ killing selection composed of many devices with different catalog numbers</td></tr><tr><td>Incubator with CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sanyo</td><td>MCO15A</td><td></td></tr><tr><td>Incubator without CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Heraeus instrument</td><td>BB6220</td><td></td></tr><tr><td>Laminar flow hood PSM1200NF</td><td>Fisher&amp;nbsp;</td><td>52010120</td><td></td></tr><tr><td>DMEM medium</td><td>sigma</td><td>D5796</td><td></td></tr><tr><td>FBS gold</td><td>GE Healthcare</td><td>A15-151</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>Trypsin 10x</td><td>PAA</td><td>L11-003</td><td></td></tr><tr><td>Atomic Absorption spectrometer with Zeeman furnace system</td><td>Thermo Scientific</td><td>ICE 3500 GFZ</td><td></td></tr><tr><td>LiCl</td><td>sigma</td><td>L4408</td><td></td></tr><tr><td>Nitric Acid</td><td>sigma</td><td>438073</td><td></td></tr><tr><td>Fluorescence videomicroscopy set</td><td>Leica</td><td></td><td>Composed of many devices with different catalog numbers</td></tr><tr><td>Inverted automated microscope</td><td>Leica</td><td>DMI6000B</td><td></td></tr><tr><td>microscope stand</td><td>leica</td><td>11888906</td><td></td></tr><tr><td></td><td></td><td>11888911</td><td></td></tr><tr><td></td><td></td><td>11505180</td><td></td></tr><tr><td></td><td></td><td>11888377</td><td></td></tr><tr><td>incident fluorescence</td><td>leica</td><td>11888901</td><td></td></tr><tr><td></td><td></td><td>11504166</td><td></td></tr><tr><td>motor bracket</td><td>leica</td><td>11888379</td><td></td></tr><tr><td></td><td></td><td>11505234</td><td></td></tr><tr><td></td><td></td><td>11521505</td><td></td></tr><tr><td></td><td></td><td>11522106</td><td></td></tr><tr><td>LED transmission light&amp;nbsp;</td><td>leica</td><td>8097321</td><td></td></tr><tr><td></td><td></td><td>8102034</td><td></td></tr><tr><td></td><td></td><td>11521580</td><td></td></tr><tr><td>motorized plate</td><td>leica</td><td>11522068</td><td></td></tr><tr><td></td><td></td><td>11531172</td><td></td></tr><tr><td></td><td></td><td>11521734</td><td></td></tr><tr><td></td><td></td><td>11521719</td><td></td></tr><tr><td></td><td></td><td>11888423</td><td></td></tr><tr><td></td><td></td><td>11888424</td><td></td></tr><tr><td>camera output</td><td>leica</td><td>11888373</td><td></td></tr><tr><td></td><td></td><td>11507807</td><td></td></tr><tr><td></td><td></td><td>11888393</td><td></td></tr><tr><td></td><td></td><td>11888259</td><td></td></tr><tr><td></td><td></td><td>11888258</td><td></td></tr><tr><td></td><td></td><td>11541510</td><td></td></tr><tr><td>images acquisition/analysis software</td><td>leica</td><td>11888375</td><td></td></tr><tr><td>optics</td><td>leica</td><td>11506507</td><td></td></tr><tr><td></td><td></td><td>11506243</td><td></td></tr><tr><td></td><td></td><td>11506203</td><td></td></tr><tr><td>fluorescence Xenon lamp</td><td>leica</td><td>DMI6000</td><td></td></tr><tr><td>camera</td><td>hamamatsu</td><td>8100601</td><td></td></tr><tr><td>metafluor/Mmfluor software</td><td></td><td>11640905</td><td></td></tr><tr><td>pH sensitive probe, BCECF-AM</td><td>life technologies</td><td>B1170</td><td></td></tr><tr><td>Nigericin</td><td>Sigma</td><td>N7143</td><td></td></tr></tbody></table>

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Selection:
The H+ killing selection is based on the diffusion of the ammonium weak base, as depicted in Figure 1A. The effect of weak bases and acids diffusion on intracellular pH has been pioneered by Walter Boron and collaborators22. The elegant idea to use this phenomenon to produce a lethal acidification for positive genetic selection was then developed by Jacques Pouyss&#233;gur17. Under such a protocol, the intracellular pH drops to about...

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Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

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Biology

9.2K Views.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Video: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Biochemistry

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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.

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.

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 ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

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