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

Summary

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.

Abstract

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.

Introduction

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 genes^3-6.

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 Syndromes^8-9, family-based autism¹⁰ and Attention Deficit Hyperactivity Disorder^11-12. 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, Pouysségur 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.

Protocol

1. H⁺ Killing Selection

1. Cell lines
   1. 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’s instructions.
2. Solutions
   1. Prepare three sterile solutions described as below. Sterilize these solutions by filtration (0.22 µm).
      1. 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.
      2. 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.
      3. 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.
3. Before starting the selection, obtain a cell culture incubator that can be used at 37 °C with no additional CO₂ from external tank (termed CO₂-“free” 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).
4. H⁺ killing procedure:
   1. Incubate the cells expressing the intracellular NHE of interest for 1 hour in the Loading Solution, at 37 °C in the CO₂-free incubator.
   2. 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.
   3. Eliminate the rinse medium by aspiration and incubate the cells for one hour in recovery solution again at 37 °C in the CO₂ free incubator.
   4. Replace the recovery medium with regular culture medium (typically DMEM with 7.5% FCS) and grow the cells in standard culture conditions (37 °C in a humidified atmosphere of 5% CO₂ and 95% air).
5. 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.
   1. 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.
6. 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).

2. Intracellular pH Measurements

2.1) Fluorescence pH Imaging

1. 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’s protocols.
   1. Alternatively, use other pH–sensitive probes, following the manufacturer’s protocols.
2. 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.
   1. If possible, use the appropriate set of filters and illumination conditions to setup comparable fluorescence values for λ1 and λ2 so that the ratio approaches 1. Also avoid basal fluorescence values that are set either saturating or too low, both for λ1 and/or λ2. 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.

2.2) Measurement of NHE Forward Activity (Extrusion of Protons from the Cytosol)

1. Acidify cells by a 1 hr incubation in the NH₄⁺ Loading solution (see step 1.4) in the absence of CO₂.
2. Add BCECF-AM (5 µM final concentration) to the solution for the last five minutes and then rinse it with NH₄⁺ loading solution to eliminate the extracellular probe.
3. 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.
4. 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

2.3) Measurement of NHE Reverse Activity

NOTE: The principle here is to invert the transmembrane ionic gradients.

1. Prior to measurement, load the cells with the intracellular cation of interest (see below).
2. Incubate them for 5 min with BCECF/AM (see step 2.2.2); rinse them, mount them on the video microscope.
3. Perfuse the loading solution and take several images to record a stable baseline.
4. 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.
5. 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.
6. 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.

2.4) Data Treatment and Calibration:

NOTE: This step applies both to the forward and reverse measurements.

1. At the end of each experiment, perfuse cells with a solution of 140 mM KCl, 20 mM HEPES and 5 µM nigericin adjusted to pH values between 6.5 and 7.4.
2. 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.
3. Calculate intracellular pH values for each individual cell or region of interest using the following equation:
   pH = pKa + (Log (R-Rmin)/(R_max-R))x F_min(λ2)/F_max(λ2)
4. Use the F_min(λ2)/F_max(λ2) 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.
5. 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:
   1. 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.

3. Measurements of Initial Rates of NHE7 by Fast Li⁺ Uptake

1. 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.
2. 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.
3. 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.
4. 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.
5. Lyse the cells in 25% Nitric acid. Apply 250 µl 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.
6. 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.
7. 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’s instructions as these machines are very precise but also quite delicate.

Results

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 Pouysségur¹⁷. Under such a protocol, the intracellular pH drops to about 5.5 ...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Standard cell culture equipement			Used for H+ killing selection composed of many devices with different catalog numbers
Incubator with CO2	Sanyo	MCO15A
Incubator without CO2	Heraeus instrument	BB6220
Laminar flow hood PSM1200NF	Fisher	52010120
DMEM medium	sigma	D5796
FBS gold	GE Healthcare	A15-151
Penicilin/streptomycin	PAA	P11-010
Trypsin 10x	PAA	L11-003
Atomic Absorption spectrometer with Zeeman furnace system	Thermo Scientific	ICE 3500 GFZ
LiCl	sigma	L4408
Nitric Acid	sigma	438073
Fluorescence videomicroscopy set	Leica		Composed of many devices with different catalog numbers
Inverted automated microscope	Leica	DMI6000B
microscope stand	leica	11888906
		11888911
		11505180
		11888377
incident fluorescence	leica	11888901
		11504166
motor bracket	leica	11888379
		11505234
		11521505
		11522106
LED transmission light	leica	8097321
		8102034
		11521580
motorized plate	leica	11522068
		11531172
		11521734
		11521719
		11888423
		11888424
camera output	leica	11888373
		11507807
		11888393
		11888259
		11888258
		11541510
images acquisition/analysis software	leica	11888375
optics	leica	11506507
		11506243
		11506203
fluorescence Xenon lamp	leica	DMI6000
camera	hamamatsu	8100601
metafluor/Mmfluor software		11640905
pH sensitive probe, BCECF-AM	life technologies	B1170
Nigericin	Sigma	N7143