and philip a. gale*
TRANSCRIPT
Supramolecular methods: the chloride/nitrate transmembrane exchange
assay
Laura A. Jowett1 and Philip A. Gale*1
1School of Chemistry, The University of Sydney, NSW 2006, Australia
The chloride/nitrate anion exchange assay is one of the most common methods of
measuring chloride transport across lipid bilayers. In this methods paper, we provide a
step-by-step procedure for using this assay based on ion selective electrodes and
highlight the limitations of this approach in assessing the transmembrane chloride
transport capability of anionophores.
Keywords: supramolecular chemistry, anion transport, ion-selective electrode, assay
Introduction
Anion transport is a well-established, and still expanding field of research due to its relevance in
biological systems.1,2 There are many membrane spanning ion-channels responsible for critical
biological processes, such as maintaining cellular pH and volume, and cellular signalling.3 Defects in
these channels can reduce the flux of anions through membranes and lead to life-limiting diseases
known as channelopathies; examples include Bartter syndrome and the more commonly known cystic
fibrosis.4-7 Small molecule anion transporters have been developed with the potential to bypass these
faulty channels and restore the transport of anions across membranes. There are various methods
available for studying anion transport, including assays that use pH sensitive fluorescent dyes, voltage
clamp measurements and NMR assays. However, one of the most widely used is the chloride/nitrate
(Cl-/NO3-) exchange ion selective electrode (ISE) assay, shown in figure 1, which is used to directly
measure chloride transport.8-10
Figure 1. A schematic of the chloride/nitrate exchange ion selective electrode assay.
The first reports of the Cl-/NO3- exchange ISE assay were in the early 2000’s11-13 and since then it has
been used for directly monitoring chloride transport.14,15 In this assay, lipid vesicles are prepared in
sodium chloride solution and, using dialysis, the salt in the external solution can be exchanged for
sodium nitrate. This provides a vesicle suspension with a chloride concentration gradient between the
intra- and extra-vesicular solutions allowing anionophores to facilitate transport passively down the
gradient. The assay relies on the fact that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
lipid bilayers are impermeable to chloride in the absence of an anionophore. Chloride efflux mediated
by a transporter is detected using a chloride selective electrode in the external solution, whilst back
transport of nitrate dissipates the charge gradient created by chloride transport and results in overall Cl-
/NO3- exchange. This assay provides direct detection of the chloride anion, allowing unambiguous
determination of chloride transport. The back transport of nitrate is assumed to be facile as it is more
lipophilic than chloride,16 however caution should be displayed when interpreting the results obtained
as it has been shown that nitrate transport can be the rate-limiting transport step for some classes of
anion transporters.17 Therefore, it is best to use a combination of assays to determine relative transport
efficiencies: the Cl-/NO3- exchange ISE assay to show direct chloride transport, and uniport assays to
measure the transport of one anion alone.
Many classes of compounds have been tested in the Cl-/NO3- exchange ISE assay demonstrating its
versatility. The Davis group have reported cholapod anion transporters, a series of compounds
comprising a cholic acid steroidal scaffold with appended anion binding groups.18 The first transporters
were tested in the Cl-/NO3- exchange ISE assay, with the best transporter 1 being shown in figure 2.12,19
As the compounds increased in complexity they became highly lipophilic resulting in poor aqueous
solubility.20 This solubility issue led to the development of alternative assays but highlighted a major
benefit of the Cl-/NO3- exchange ISE assay; the intrinsic deliverability test. The ability of the transporter
to be delivered to vesicles in aqueous solution is monitored; an important consideration when designing
potential therapeutics. Our group has used the Cl-/NO3- exchange ISE assay extensively for a wide range
of small molecule chloride transporters, including urea and thiourea tren-based compounds 2a and 2b,
figure 2,21,22 squaramide and thiosquaramide compounds,23,24 ortho-phenylenediamine compounds,25,26
and indole-based anionophores.27-29
Figure 2. Two compounds tested in the Cl-/NO3- exchange ISE assay. The cholapod anion transporter
(1) that achieved the highest percentage chloride efflux. One example of a (thio)urea tren based compound (2) that was tested in the Cl-/NO3
- exchange ISE assay.
An additional benefit of the Cl-/NO3- exchange ISE assay is the flexibility of conditions that can be
employed when studying chloride transport. The pH of the external solution can be tuned using a range
of buffer solutions allowing the transport capabilities to be determined at different pH. One study
involving phenylthiosemicarbazones showed that at pH 7.2 there was no chloride transport, but at pH
4.0 protonation of the transporter occurs resulting in a conformational change that allows the compound
to facilitate chloride transport.30 Azobenzene-based chloride transporters are another class of
compounds tested using the Cl-/NO3- exchange ISE assay. The transport was initially tested with the cis
and trans form separately. The closed cis isomer showed transport, whereas the open trans isomer did
not. In a subsequent experiment, in situ irradiation of the trans isomer induced a conformational change
to the cis isomer, switching the chloride transport on.31
Here, we give an overview of the experiments we perform, the equipment we use and a step-by-step
guide of how to conduct vesicle-based experiments in the Cl-/NO3- exchange ISE assay.
Experiments
The Cl-/NO3- exchange ISE assay is an excellent tool to study ion transport under varying pH conditions
as it can be easily modified by changing the internal and external buffer solutions to perform
experiments over a pH range. Small molecule ion transporters that can switch transport on or off
depending on pH could mimic the function of acid-sensing ion channels.32 Alternatively, tuning the pH
at which transport occurs has the potential to target ion transport in specific cell types or cellular
organelles. We have reported three classes of pH switchable anion transporters, the thiosquaramides,24
the oxothiosquaramides33 (both in collaboration with Jolliffe), and the phenylthiosemicarbazones 3 –
5.30 In these examples the Cl-/NO3- exchange ISE assay was run at pH 7.2 using sodium phosphate
buffered (5 mM) solutions and then compared to the results from the Cl-/NO3- exchange ISE assay tested
at pH 4.0 using sodium citrate buffered (5 mM) solutions. In all cases, significantly less chloride
transport was observed in near neutral conditions but upon repeating the experiments at an acidic pH, a
high percentage chloride efflux was observed, results for phenylthiosemicarbazones 3 – 5 are shown in
figure 3. One potential use for this family of compounds could be in targeting transporters to acidic
cellular organelles such as lysosomes. We have also explored the possibility of tuning the pKa of a
protonatable nitrogen atom in the perenosins,28,29 and probed the effect subtle differences in the pH
environment have on the chloride transport abilities. Here, the Cl-/NO3- exchange ISE assay was run at
pH 4.0 and 4.5 using citrate buffered (5 mM) solutions, where the higher degree of protonation resulted
in high chloride transport efficiencies. Poor chloride transport was observed under basic (pH 10
ethanolamine (5 mM) buffer and pH 8.2 sodium phosphate (5 mM) buffer) conditions, due to the lack
of protonation and subsequent chloride binding. As the pH decreased from pH 7.2 to 6.5 (both buffered
with sodium phosphate (5 mM)), or pH 7.2 to 6.2 (buffered with piperazine (5 mM)) the chloride
transport increased for all compounds. Compounds 6 – 8 showed the largest increase in transport, results
are shown in figure 3, and so display the best potential for targeting chloride transport in cancer cells
(external pH 6.734) over healthy cells (external pH 7.2-7.535).
Figure 3. Results of the Cl-/NO3- exchange ISE assay when testing in different pH environments. Left
– Phenylthiosemicarbazone compounds (3 - 5) show a large switch on in chloride transport in acidic environments, vesicle experiments compared at pH 7, open symbols, and pH 4, closed symbols. Right – Perenosin compounds (6 – 8) show an increase in chloride transport in slightly acidic environments, vesicle experiments compared at pH 7.2, open symbols and pH 6.5, closed symbols.
Chloride, bicarbonate and phosphate are the most abundant anions in the body,8 but other anions, such
as fluoride,36 sulfate37 and nitrate,38 and cations, such as sodium, potassium, magnesium and calcium,39
are biologically relevant. A major benefit of the ISE assay is its ease of modification, meaning it is
applicable for a range of transport studies, and can incorporate a large array of positively and negatively
charged species. Altering the internal and external salt solutions and/or using the corresponding ISE are
both ways to change the assay to study other systems. Recently, we have shown using a fluoride
selective electrode that a modified ISE assay can be employed to directly monitor fluoride transport.40
Sulfate has a high hydration energy41 and therefore poses a challenge for neutral receptors to bind and
transport across lipid bilayers. For this reason it has been used as an external, “un-transportable” counter
ion in the Cl-/HCO3- exchange ISE assay. The Cl-/HCO3
- exchange ISE assay is prepared by
encapsulating sodium chloride inside vesicles and then suspending them in an external solution of
sodium sulfate. The transporter is added to the vesicle suspension at t = 0 s and initially, negligible
chloride transport is observed. Addition of a bicarbonate pulse at t = 120 s allows Cl-/HCO3- exchange
to occur, which increases the external chloride concentration and is subsequently detected by chloride
ISE. From the Cl-/HCO3- exchange ISE assay, our group has shown a wide variety of compounds are
capable of Cl-/HCO3- exchange, including urea and thiourea tren-based compounds,21 ortho-
phenylenediamines25 and squaramides.23,42 In one particular study,22 with tren-based compounds, a high
degree of background transport was observed from t = 0 s to t = 120 s (i.e. before the addition of sodium
bicarbonate) resulting from Cl-/SO42- exchange. This led to further investigations into sulfate transport
and the development of a Cl-/SO42- exchange assay to monitor chloride efflux. Studies using this assay
were conducted to show (thio)urea tren-based, and strapped peptide compounds were capable of Cl-
/SO42- exchange whilst a 33S NMR assay was used to directly monitor sulfate transport.43
Some transporters are capable of facilitating ion pair transport and, when the compounds display good
activity in the Cl-/NO3- exchange ISE assay, it can subsequently be used to probe this activity. Using
NaCl, KCl, RbCl and CsCl as the internal solution and NaNO3 as the external solution, the assay was
tested with the strapped calixpyrroles; which showed ion pair transport only in the presence of CsCl.44
This experiment has also been used to rule out the possibility of M+/Cl- symport for a number of
compounds.25,45
Comparison of the anion transport ability between different classes of transporter, or the same class of
transporter with different anions, is important and can be achieved through quantifying the extent of
anion transport by obtaining EC50 values (the concentration of transporter needed to achieve 50%
chloride efflux in a set time period). Dose-response studies are performed by measuring the increase in
external chloride, using an ISE, over time at different transporter concentrations. Hill analysis46 is then
conducted to afford an EC50 value and a Hill coefficient, n, which is indicative of the stoichiometry of
the transporting complex. Results of dose response studies and subsequent Hill analysis for tren based
thiourea 9 can be found in figure 4.17 These studies have been performed routinely for a large variety
of compounds including urea and thiourea tren-based compounds,22 N,N′-
(phenylmethylene)dibenzamide compounds47 and acylthiourea compounds.48
Figure 4. Results of the Cl-/NO3- exchange ISE assay
from an alkyl thiourea tren-based compound (9) at different concentrations. Left – Dose response studies. Right –
Hill analysis of the data affording an EC50 value (k) and Hill coefficient (n).
Equipment
Before starting an anion transport assay, there are some essential equipment and materials required to
perform vesicle-based experiments.
1. The lipid
The choice of lipid is an important consideration as lipids have diverse properties which can affect the
transport experiments. In 2015 we performed a study using the Cl-/NO3- exchange ISE assay and found
that the chloride transport rate varied for four lipids tested; these were POPC, 10, see figure 5, 1-
palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphoethanolamine (POPE), and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The chloride
transport abilities in DPPC vesicles were measured at a higher temperature due to the higher phase
transition temperature of DPPC, and vesicles of a 3:1 POPE:POPC ratio were used. POPE had to be employed
as a lipid mixture because alone it preferentially forms multilamellar vesicles which contain different lipid
phases in the same vesicle. In this particular study the chloride transport rates in POPC and DPPC vesicles
were the same (tests performed at different temperatures), in POPG vesicles the rate decreased, and in
mixed POPE/POPC vesicles the rate increased compared to POPC.49 It should be noted that these results
might be different if the temperature and lipid compositions were changed. The study concluded that
POPC was most suitable for vesicle-based experiments due to the moderate transport rates and the
reproducibility of results.
The lipid chosen for vesicle-based transport experiments should be 1) easy to work with in the
laboratory, 2) produce repeatable results, 3) within the budget allocated, and most importantly 4) mimic
biological membranes of interest (especially if the research involves developing and testing novel
therapeutics). In the Gale group, we have used POPC from Corden Pharma and Avanti Polar Lipids for
many years,22,30,48 the A.P. Davis group has also used POPC from Avanti Polar Lipids.50-52 The Matile
group has used egg yolk phosphatidylcholine (EYPC) from Avanti Polar Lipids,53-55 and more recently
the Talukdar group has used EYPC from the same supplier.56-58 These lipids are chosen because they
mimic mammalian phospholipid composition (where POPC is the main component) and hence provide
a suitable model membrane for transport experiments. EYPC is a natural lipid mixture, with POPC
being the main component.59 The EYPC mixture contains lipids that have different aliphatic tail chains
(specific information can be found on the supplier website). Within the purchased product a variety of
tail lengths and different tail chain saturations are found; and this can therefore result in slightly different
compositions between purchased batches and an average molecular weight of the lipid. POPC is a
synthetic lipid product with certified purity (see supplier website for more information). This ensures
that the purchased synthetic lipid is consistent between batches and so gives reliable experimental
results.60 Furthermore, we have found that the free fatty acid content can vary with different suppliers
of lipid and, although this does not affect the Cl-/NO3- exchange ISE assay, it can have consequences in
other transport assays.61
Figure 5. Structure of commonly used POPC lipid for vesicle-based anion transport assays.
2. The salts
Anion transport experiments can focus on anion exchange, anion uniport, or osmosis accompanying
salt transport. In all these cases buffer solutions are required for the internal and external solutions of
the vesicles. The salts we use for preparing the buffer solutions are analytical grade chemicals with high
purity, usually ≥ 99 %.
Standard assay conditions have been developed by our group for the Cl-/NO3- exchange ISE assay. We
have used sodium phosphate (5 mM) buffer for experiments conducted at pH 7.2. This is made up of
the acidic and basic form of sodium phosphate, sodium dihydrogen phosphate dihydrate and disodium
hydrogen phosphate. The internal solution contains sodium chloride (487 mM) and the external solution
contains sodium nitrate (487 mM). The total ionic strength of the internal and external solutions must
be kept constant throughout the duration of an experiment. For experiments conducted at an acidic pH,
a citrate or piperazine buffer solution is used, and for experiments performed at a basic pH either
tris(hydroxymethyl)aminomethane or ethanolamine buffer solution is used.
3. The ion selective electrode
ISEs have long been used to measure ion concentration and activity in aqueous solution.62 Some benefits
include that they are simple to use, they are very robust when properly cared for and can be used for a
wide range of applications, such as analysis of water, soil, and dairy products. However, there are
limitations to note, the electrodes do tend to have a limited shelf life, and signal interference can occur
with certain ions, such as CN- and S- if they are present as contaminants in the test solution (check the
electrode specification for more details).63,64 There are several types of ISE available with different
sensor types and operations.65 Solid state electrodes, where the electrode potential is measured across a
solid, polished crystalline membrane, have a long life time, rapid response times and minor maintenance
requirements. One example of this is the Fisherbrand accumet Chloride Combination Electrode66 we
use in the Gale group to detect chloride efflux in vesicle experiments.
Chloride selective electrodes vary depending on the manufacturer, therefore the user manual for the
electrode should be consulted before use.67,68 The combination chloride solid state electrode our group
uses contains a sensing element, the membrane, which develops an electrode potential when in contact
with the test solution containing chloride. The potential, which is dependent on the free chloride ions in
the solution, is measured against a reference with a digital pH/mV meter or a specific ion meter (we use
an Orion Star Conductivity Benchtop Meter69). The reference electrode, found either internally in the
case of combination electrodes or externally in the case of half cell electrodes, contains a known and
fixed concentration of the ion of study. The Nernst equation is used to describe the measured potential
which corresponds to the chloride ions in the test solution:
E = E0 + S log(A)
Where E is the measured electrode potential, E0 is the reference potential, S is the electrode slope and
A is the chloride ion activity level in solution. Often electrode calibration is required before use which
allows E0 and S to be determined, again consult the user manual for more information on the electrode.
The chloride ion activity is related to the free chloride ion concentration by the activity coefficient:
A = γ Cf
Where A is the chloride ion activity level in solution, γ is the activity coefficient and Cf is the free
chloride ion concentration. Activity coefficients are usually dependent on the total ionic strength but
when the background ionic strength is high and constant, relative to the sensed ion concentration (which
is the case for buffer solutions prepared in the same way as described in section 2. The Salts), the activity
coefficient is constant and therefore directly proportional to the chloride ion concentration.
Over the course of an experiment the electrode potential is recorded every 5 s with a digital pH/mV
meter (see above) connected to a PC. At the end of an experiment the recorded electrode potentials are
converted into chloride concentrations (C) using the equations described above. These concentrations
are then normalised:
Cf = C – C0
Where Cf is the final concentration, C is the calculated concentration at each electrode potential reading
and C0 is the initial concentration. Normalisation then allows a percentage chloride efflux to be
calculated at the end of each experiment.
4. Other things to think about.
As well as the important materials detailed in the above sections, there is some other equipment required
for vesicle-based anion transport experiments. Drying the lipid requires a rotary evaporator and a high
vacuum pump to ensure a smooth lipid film is created. An analytical balance is used to obtain the exact
weight of the lipid and for weighing the salts for the buffer solutions. Preparing the internal and external
buffer solutions requires access to ultrapure, Type 1 (also known as Milli-Q) water to prevent ion
contamination. A solution of Triton X-100 (11 w%, in H2O:DMSO 7:1 v:v) is readily required for lysing
the vesicles in order to calibrate the 100% chloride efflux value. Liquid nitrogen, an extrusion kit
including 200 nm polycarbonate membranes and dialysis tubing are essential for the vesicle preparation
steps. Our group also uses stopwatches, micropipettes, micro PTFE stirrer bars and single use glass
vials (10 – 20 mL), that fit the electrode in with a small amount of space for a pipette tip, when
performing the experiments. An example of the experimental set up is shown in figure 6.
Figure 6. An example of the experimental set up used by the Gale group when running a Cl-/NO3-
exchange ISE assay.
Procedure
Below is a general procedure, as used by the Gale group for preparing vesicles and running a Cl-/NO3-
exchange ISE assay at pH 7.2.
Step-by-step Solution Preparation
1. Prepare the buffer solution. Dissolve sodium dihydrogen phosphate dihydrate (0.294 g, 0.0019
mol) and disodium hydrogen phosphate (anhydrous) (1.150 g, 0.0081 mol) in ultrapure, Type
1 water (2 L) in a beaker (Figure 7).
2. Prepare the internal solution. Dissolve sodium chloride (2.846 g, 0.0487 mol) in the phosphate
buffer solution (100 mL) and when fully dissolved carefully adjust the pH to 7.20 if necessary
– using HCl to decrease the pH, or NaOH solution to increase the pH.
3. Prepare the external solution. Dissolve sodium nitrate (78.632 g, 0.9251 mol) straight into the
remaining phosphate buffer solution (1900 mL) and when fully dissolved carefully adjust the
pH to 7.2 – using HCl or NaOH solution to adjust the pH as required.
Step-by-step Vesicle Preparation
1. Dissolve POPC (1 g) in chloroform (35 mL), resulting in a 37.5 mM solution.
2. Weigh a round bottom flask and add between 0.5 – 4 mL of the chloroform solution using a
volumetric pipette (remember how much you add as this will be used in step 6), depending on
the number of experiments to run (approximately 0.15 mL of lipid per experiment to run).
3. Remove the organic solvent gently on a rotary evaporator, creating a smooth lipid film. Ensure
the flask is rotating slowly and reduce the pressure gradually until chloroform begins to
evaporate (Figure 8).
4. Dry the lipid film in a vacuum desiccator for a minimum of 6 hrs, preferably overnight.
5. Weigh the round bottom flask and lipid film, this is essential for calculating the concentration
of lipid.
6. Using an autopipette add the same volume of internal solution as the lipid chloroform solution
used in step 2.
7. Ensure the lipid is fully hydrated by vortexing the solution for 5 minutes, or until there is no
lipid remaining on the interior of the flask (Figure 9).
8. Perform nine freeze-thaw steps. Fully freeze the lipid suspension in liquid nitrogen (invert to
check all the solution is frozen), then defrost in lukewarm water and repeat. Dry the outside of
the round bottom flask before placing in liquid nitrogen (Figure 10).
9. Allow the lipid solution to rest for 30 minutes at room temperature.
10. During this time wash all parts of the extrusion kit with ethanol then water. Repeat the washing
three times and then assemble the extrusion kit with a 200 nm polycarbonate membrane.
Instructions can be found on the Avanti lipid website for help with the assembly.70
11. Set aside some of the external sodium nitrate solution (300 mL approx.) to be used later. This
can be stored in a sealed jar in a fridge for approximately 1 week, note that the solution should
be equilibrated to room temperature before opening.
12. Cut a piece of dialysis tubing to the appropriate length (5 cm per 1 mL), place a buoyant clip
on one end and put the tubing in the remaining external sodium nitrate solution (1600 mL
approx.) leaving it to stir.
13. Fill one of the syringes with 1 mL of the lipid solution and place it into one side of the extrusion
kit. Leaving the other syringe empty, place it in the opposite side. Ensure the syringe needle
tips are well tightened to prevent leakage.
14. Push the lipid solution back and forth through the extrusion kit twenty-five times then place the
extruded lipid solution in a vial, recording the volume. Repeat the extrusions using the same
syringe for the unrefined lipid and extruded lipid each time and combine the remaining lipid
(Figure 11) . Clean the extrusion kit with ethanol and then water.
15. Take the wetted dialysis tubing out of the external solution using tweezers, dry slightly on some
lab paper and then open the tubing with tweezers.
16. Whilst holding the tubing open with tweezers, carefully pipette the extruded lipid solution into
the dialysis tubing and close the top with a second buoyant clip.
17. Place the clipped dialysis tubing containing the extruded lipid solution into the external solution
and leave stirring gently for a minimum of two hours, preferably overnight. The duration of
this dialysis step depends on the pH at which experiments are to be performed. For vesicles
being tested at acidic pH, pH 4 to pH 6, this should be 2hrs, at all other pH dialysis can be left
overnight
18. After dialysis, carefully remove the tubing containing the lipid from the external solution using
tweezers and transfer the lipid solution into a volumetric flask (10 mL).
19. Top up the volumetric flask with the external solution set aside in step 11 and transfer the final
lipid solution to a capped vial.
20. Calculate the concentration of lipid in your vial, see calculations below:
Mass of lipid (g) = Mass recorded in Step 5 (g) – Mass recorded in Step 2 (g)
Moles of lipid (mol) = Mass of lipid (g) / Molecular weight of lipid (gmol-1) (760.09 gmol-1 for
POPC)
Concentration of lipid (moldm-3) = Moles of lipid (mol) / Volume of volumetric flask (L) (0.010 L)
21. Use this concentration to calculate how much lipid to use in each experiment. The desired lipid
solution has a concentration of 1 mM in 5 mL, see calculations below:
Volume of lipid for experiment (L) = (Desired concentration (0.001 M) x Final solution volume
(0.005 L)) / Lipid concentration (moldm-3)
Step-by-step Experimental Procedure
1. Prepare the electrode; in general this requires a top up of the internal electrode solution, then to
wash the electrode with water, ethanol and water, then calibrate the electrode. See the
instruction manual for more specific instructions for each electrode.
2. Prepare a known concentration DMSO solution (0.5 mL approx.) of the transporter to be tested.
Here it is best to make a concentrated stock solution (around 50 mM) that can be diluted for
subsequent measurements.
3. Prepare the experimental solution by adding the calculated volume of lipid for the experiment
(step 21 of vesicle preparation) using a micropipette to a single use glass vial equipped with a
clean micro PTFE stirrer bar. Make up the solution to 5 mL by adding the external solution
(prepared in step 3 of solution preparation and set aside in step 11 of vesicle preparation) using
a micropipette.
4. Place the electrode in the experimental solution and set the stirring to 400 rpm.
5. Prepare to begin the experiment; set the stopwatch and/or the electrode reader to zero
(depending on the equipment). Ensure the electrode reader is set to record every 5 s for the
duration of the experiment. Experiments are usually run for 300 s, but this time can be altered
to best suit the transporter being tested.
6. Simultaneously start the stopwatch and/or the electrode reader and, with the pipette tip slightly
submerged in the solution, add DMSO (10 µL) using a micropipette; this will be the control
run.
7. Allow the experiment to proceed, ensuring the electrode reader is recording. The reading on the
electrode should not change because DMSO does not facilitate chloride transport.
8. At 300 s (or at the time determined to end the experiment) add the Triton X-100 detergent
solution (50 µL) to lyse the vesicles.
9. Leave the experiment running for an additional 120 s to allow the 100% chloride efflux reading
to settle, then stop the electrode reader and stopwatch.
10. Remember to save your data.
11. At the end of the experiment dispose of the glass vial and the vesicle solution in the appropriate
waste, wash the micro PTFE stirrer bar with water, ethanol and water and dry for the next
experiment, and wash the electrode with water, ethanol and water and dry for the next
experiment.
12. Repeat steps 3 to 11, adding the transporter solution (10 µL) in place of DMSO in step 6, and
watching the reading electrode decrease as chloride is transported out of the vesicles into the
external solution in step 7. Use all the lipid for running experiments (the lipid should be used
in the same day). The same transporter can be tested at a variety of concentrations to form a
dose-response curve allowing Hill analysis, or many different transporters can be tested in the
same conditions to compare the transport ability of a series of transporters.
Photographs
Figure 7. Solution preparation step 1. Accurately weighing out salts for the buffer solution.
Figure 8. Vesicle preparation step 3. Removing the organic solvent gently on a rotary evaporator.
Figure 9. Vesicle preparation step 7. Vortexing the lipid solution to ensure it is fully hydrated.
Figure 10. Vesicle preparation step 8. Left – Fully freezing the lipid solution in liquid nitrogen. Right –
Defrosting the lipid solution in tepid water.
Figure 11. Vesicle preparation step 13/14. Extruding the lipid.
What to do if…
1. You overshoot the pH when adding acid or base during adjustment (step 2 and 3 of solution
preparation)
If this happens you can use the alternate acid/base to return the pH to 7.2. It should be noted
this is not desirable and should be avoided as it can change the ionic strength of the solution.
2. You forget to weigh the flask (step 2 and 5 of vesicle preparation)
If this happens you cannot calculate the concentration of the lipid, meaning you cannot
determine the ratio of lipid to transporter and therefore the experimental results for that batch
of lipid are void.
3. You forget to separate some of the external solution (step 11 of vesicle preparation)
If this happens and you use all the prepared external solution for dialysis, you have to make
another phosphate buffered sodium nitrate solution to use as the external solution for your
experiments.
4. You lose some of the lipid during the extrusion processes (step 13 and 14 of vesicle preparation)
If this happens you should record the volume of lipid obtained after the extrusion process and
use it to calculate the moles of lipid after extrusion, shown below:
Mass of lipid (g) = Mass recorded in Step 5 (g) – Mass recorded in Step 2 (g)
Moles of lipid (mol) = Mass of lipid (g) / Molecular weight of lipid (gmol-1) (760.09 gmol-1 for
POPC)
Moles of lipid after extrusion (mol) = (Volume of lipid in chloroform solution in Step 2 (mL) /
Volume of lipid after extrusion (mL)) x Moles of lipid (mol)
Concentration of lipid (moldm-3) = Moles of lipid after extrusion (mol) / Volume of volumetric
flask (L) (0.010 L)
5. Your transporter is not soluble in DMSO (step 2 of experimental procedure)
If this is the case, you can dissolve your compound in any solvent the compound is soluble in
as long as the solvent is miscible with water.
6. You forget to start the stopwatch/electrode reader at the beginning of an experiment (step 6 of
experimental procedure)
If this happens you are not recording the electrode reading over the course of the experiment.
It is also unclear when the experiment began and so you cannot determine when to lyse the
vesicles and when to take the final reading. Therefore, the results from this experimental run
are void.
7. Your DMSO run shows significant chloride efflux (step 6 of experimental procedure)
If this happens you should clean the PTFE stirrer bar and the electrode very well before setting
up a new experimental run to remove any chance of contamination with other compounds. If
after the second blank run there is still significant chloride efflux your vesicles might be leaky
and so are unsuitable for subsequent experiments.
8. Your transporter crashes out when you add it to the vesicle suspension (step 6 (repeated) of
experimental procedure)
If this happens it is likely that your transporter has low solubility in aqueous solution and does
not partition quickly into the membrane. If you used a high concentration DMSO solution of
transporter you can dilute the solution and try at a lower concentration (between 5 mM – 0.5
mM). If it continues to be insoluble at lower concentrations then preincorporation of your
transporter is suggested.19
9. You forget to lyse the vesicles at 300 s or at the time determined to end the experiment (step 8
of experimental procedure)
If this happens and you leave the experiment running for longer than the specified time, the
experiment is no longer consistent and so results could become inconsistent. Therefore, the
results from this experimental run are void.
10. You leave the experiment running for longer than 120 s after the addition of detergent (step 9
of experimental procedure)
If this happens then stop the experiment. For consistency in the results you should set a time
point (at least 120 s, but could be greater) at which you record the final chloride concentration
for all experimental runs.
11. You forget to save your data (step 10 of experimental procedure)
If this happens you will have to repeat the experimental run and then save the data.
12. You cannot get repeatable results
If this happens there could be some interference of your compound with the electrode. This is
sometimes the case at high concentrations, so you could dilute the sample and try at a lower
transporter concentration. It could potentially be due to the electrode, so check the age of the
electrode, make sure, if suitable, the internal solution is filled to the top, and clean the
membrane. This could also be due to poor aqueous solubility, see point 8 of frequently asked
questions
Acknowledgements
LAJ thanks The University of Sydney for a postgraduate scholarship. PAG thanks The University of
Sydney and the ARC (DP180100612) for funding.
References
(1) Busschaert, N.; Gale, P. A. Small-Molecule Lipid-Bilayer Anion Transporters for Biological Applications. Angew. Chem. Int. Ed. 2013, 52, 1374-1382.
(2) Gale, P. A.; Perez-Tomas, R.; Quesada, R. Anion Transporters and Biological Systems. Acc. Chem. Res. 2013, 46, 2801-2813. (3) Gadsby, D. C. Ion channels versus ion pumps: the principal difference, in principle. Nat. Rev. Mol. Cell. Biol. 2009, 10, 344. (4) Ashcroft, F. M.: Ion Channels and Disease; Academic Press: San Diego, 2000. (5) Vankeerberghen, A.; Cuppens, H.; Cassiman, J.-J. The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions. J. Cyst. Fibros. 2002, 1, 13-29. (6) Miyamura, N.; Matsumoto, K.; Taguchi, T.; Tokunaga, H.; Nishikawa, T.; Nishida, K.; Toyonaga, T.; Sakakida, M.; Araki, E. Atypical Bartter Syndrome with Sensorineural Deafness with G47R Mutation of the β-Subunit for ClC-Ka and ClC-Kb Chloride Channels, Barttin. J. Clin. Endocrinol. Metab. 2003, 88, 781-786. (7) Ashcroft, F. M. From molecule to malady. Nature 2006, 440, 440. (8) Davis, A. P.; Sheppard, D. N.; Smith, B. D. Development of synthetic membrane transporters for anions. Chem. Soc. Rev. 2007, 36, 348-357. (9) Davis, J. T.; Okunola, O.; Quesada, R. Recent advances in the transmembrane transport of anions. Chem. Soc. Rev. 2010, 39, 3843-3862. (10) Benke, B. P.; Aich, P.; Kim, Y.; Kim, K. L.; Rohman, M. R.; Hong, S.; Hwang, I.-C.; Lee, E. H.; Roh, J. H.; Kim, K. Iodide-Selective Synthetic Ion Channels Based on Shape-Persistent Organic Cages. J. Am. Chem. Soc. 2017, 139, 7432-7435. (11) Schlesinger, P. H.; Ferdani, R.; Liu, J.; Pajewska, J.; Pajewski, R.; Saito, M.; Shabany, H.; Gokel, G. W. SCMTR: A Chloride-Selective, Membrane-Anchored Peptide Channel that Exhibits Voltage Gating. J. Am. Chem. Soc. 2002, 124, 1848-1849. (12) Koulov, A. V.; Lambert, T. N.; Shukla, R.; Jain, M.; Boon, J. M.; Smith, B. D.; Li, H.; Sheppard, D. N.; Joos, J.-B.; Clare, J. P.; Davis, A. P. Chloride Transport Across Vesicle and Cell Membranes by Steroid-Based Receptors. Angew. Chem. Int. Ed. 2003, 42, 4931-4933. (13) Koulov, A. V.; Mahoney, J. M.; Smith, B. D. Facilitated transport of sodium or potassium chloride across vesicle membranes using a ditopic salt-binding macrobicycle. Org. Biomol. Chem. 2003, 1, 27-29. (14) Spooner, M. J.; Gale, P. A. A tripodal tris-selenourea anion transporter matches the activity of its thio- analogue but shows distinct selectivity. Supramol. Chem. 2018, 1-6. (15) Jowett, L. A.; Howe, E. N. W.; Wu, X.; Busschaert, N.; Gale, P. A. New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(thio)ureas. Chem. Eur. J. 2018, 24, 10475-10487. (16) Valkenier, H.; Davis, A. P. Making a Match for Valinomycin: Steroidal Scaffolds in the Design of Electroneutral, Electrogenic Anion Carriers. Acc. Chem. Res. 2013, 46, 2898-2909. (17) Yang, Y.; Wu, X.; Busschaert, N.; Furuta, H.; Gale, P. A. Dissecting the chloride-nitrate anion transport assay. Chem. Commun. 2017, 53, 9230-9233. (18) Davis, A. P.; Perry, J. J.; Williams, R. P. Anion Recognition by Tripodal Receptors Derived from Cholic Acid. J. Am. Chem. Soc. 1997, 119, 1793-1794. (19) McNally, B. A.; Koulov, A. V.; Lambert, T. N.; Smith, B. D.; Joos, J.-B.; Sisson, A. L.; Clare, J. P.; Sgarlata, V.; Judd, L. W.; Magro, G.; Davis, A. P. Structure–Activity Relationships in Cholapod Anion Carriers: Enhanced Transmembrane Chloride Transport through Substituent Tuning. Chem. Eur. J. 2008, 14, 9599-9606. (20) Judd, L. W.; Davis, A. P. From cholapod to cholaphane transmembrane anion carriers: accelerated transport through binding site enclosure. Chem. Commun. 2010, 46, 2227-2229. (21) Busschaert, N.; Gale, P. A.; Haynes, C. J. E.; Light, M. E.; Moore, S. J.; Tong, C. C.; Davis, J. T.; Harrell, J. W. A. Tripodal transmembrane transporters for bicarbonate. Chem. Commun. 2010, 46, 6252-6254.
(22) Busschaert, N.; Wenzel, M.; Light, M. E.; Iglesias-Hernández, P.; Pérez-Tomás, R.; Gale, P. A. Structure–Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination. J. Am. Chem. Soc. 2011, 133, 14136-14148. (23) Busschaert, N.; Kirby, I. L.; Young, S.; Coles, S. J.; Horton, P. N.; Light, M. E.; Gale, P. A. Squaramides as Potent Transmembrane Anion Transporters. Angew. Chem. Int. Ed. 2012, 51, 4426-4430. (24) Busschaert, N.; Elmes, R. B. P.; Czech, D. D.; Wu, X.; Kirby, I. L.; Peck, E. M.; Hendzel, K. D.; Shaw, S. K.; Chan, B.; Smith, B. D.; Jolliffe, K. A.; Gale, P. A. Thiosquaramides: pH switchable anion transporters. Chem. Sci. 2014, 5, 3617-3626. (25) Moore, S. J.; Haynes, C. J. E.; Gonzalez, J.; Sutton, J. L.; Brooks, S. J.; Light, M. E.; Herniman, J.; Langley, G. J.; Soto-Cerrato, V.; Perez-Tomas, R.; Marques, I.; Costa, P. J.; Felix, V.; Gale, P. A. Chloride, carboxylate and carbonate transport by ortho-phenylenediamine-based bisureas. Chem. Sci. 2013, 4, 103-117. (26) Karagiannidis, L. E.; Haynes, C. J. E.; Holder, K. J.; Kirby, I. L.; Moore, S. J.; Wells, N. J.; Gale, P. A. Highly effective yet simple transmembrane anion transporters based upon ortho-phenylenediamine bis-ureas. Chem. Commun. 2014, 50, 12050-12053. (27) Moore, S. J.; Wenzel, M.; Light, M. E.; Morley, R.; Bradberry, S. J.; Gomez-Iglesias, P.; Soto-Cerrato, V.; Perez-Tomas, R.; Gale, P. A. Towards "drug-like" indole-based transmembrane anion transporters. Chem. Sci. 2012, 3, 2501-2509. (28) Van Rossom, W.; Asby, D. J.; Tavassoli, A.; Gale, P. A. Perenosins: a new class of anion transporter with anti-cancer activity. Org. Biomol. Chem. 2016, 14, 2645-2650. (29) Jowett, L. A.; Howe, E. N. W.; Soto-Cerrato, V.; Van Rossom, W.; Pérez-Tomás, R.; Gale, P. A. Indole-based perenosins as highly potent HCl transporters and potential anti-cancer agents. Sci. Rep. 2017, 7, 9397. (30) Howe, E. N. W.; Busschaert, N.; Wu, X.; Berry, S. N.; Ho, J.; Light, M. E.; Czech, D. D.; Klein, H. A.; Kitchen, J. A.; Gale, P. A. pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones. J. Am. Chem. Soc. 2016, 138, 8301-8308. (31) Choi, Y. R.; Kim, G. C.; Jeon, H.-G.; Park, J.; Namkung, W.; Jeong, K.-S. Azobenzene-based chloride transporters with light-controllable activities. Chem. Commun. 2014, 50, 15305-15308. (32) Yoder, N.; Yoshioka, C.; Gouaux, E. Gating mechanisms of acid-sensing ion channels. Nature 2018, 555, 397. (33) Elmes, R. B. P.; Busschaert, N.; Czech, D. D.; Gale, P. A.; Jolliffe, K. A. pH switchable anion transport by an oxothiosquaramide. Chem. Commun. 2015, 51, 10107-10110. (34) Mazzio, E. A.; Smith, B.; Soliman, K. F. A. Evaluation of endogenous acidic metabolic products associated with carbohydrate metabolism in tumor cells. Cell Biol. Toxicol. 2010, 26, 177-188. (35) Bailey, K. M.; Wojtkowiak, J. W.; Hashim, A. I.; Gillies, R. J. Targeting the Metabolic Microenvironment of Tumors. Adv. Pharmacol. 2012, 65, 63-107. (36) Stockbridge, R. B.; Lim, H.-H.; Otten, R.; Williams, C.; Shane, T.; Weinberg, Z.; Miller, C. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 15289-15294. (37) Markovich, D. Physiological Roles and Regulation of Mammalian Sulfate Transporters. Physiol. Rev. 2001, 81, 1499-1533. (38) Addiscott, T. M.; Benjamin, N. Nitrate and human health. Soil Use and Management 2004, 20, 98-104. (39) Portelli, C. The role of the sodium, potassium, magnesium and calcium ions in the transfer of bioenergy, and the possibility of their substitution by other cations. Physiologie (Bucarest) 1977, 14, 43-46.
(40) Clarke, H. J.; Howe, E. N. W.; Wu, X.; Sommer, F.; Yano, M.; Light, M. E.; Kubik, S.; Gale, P. A. Transmembrane Fluoride Transport: Direct Measurement and Selectivity Studies. J. Am. Chem. Soc. 2016. (41) Marcus, Y. Thermodynamics of solvation of ions. Part 5.-Gibbs free energy of hydration at 298.15 K. J. Chem. Soc., Faraday Trans. 1991, 87, 2995-2999. (42) Busschaert, N.; Park, S.-H.; Baek, K.-H.; Choi, Y. P.; Park, J.; Howe, E. N. W.; Hiscock, J. R.; Karagiannidis, L. E.; Marques, I.; Félix, V.; Namkung, W.; Sessler, J. L.; Gale, P. A.; Shin, I. A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations. Nat. Chem. 2017, 9, 667-675. (43) Busschaert, N.; Karagiannidis, L. E.; Wenzel, M.; Haynes, C. J. E.; Wells, N. J.; Young, P. G.; Makuc, D.; Plavec, J.; Jolliffe, K. A.; Gale, P. A. Synthetic transporters for sulfate: a new method for the direct detection of lipid bilayer sulfate transport. Chem. Sci. 2014, 5, 1118-1127. (44) Yano, M.; Tong, C. C.; Light, M. E.; Schmidtchen, F. P.; Gale, P. A. Calix[4]pyrrole-based anion transporters with tuneable transport properties. Org. Biomol. Chem. 2010, 8, 4356-4363. (45) Berry, S. N.; Soto-Cerrato, V.; Howe, E. N. W.; Clarke, H. J.; Mistry, I.; Tavassoli, A.; Chang, Y.-T.; Perez-Tomas, R.; Gale, P. A. Fluorescent transmembrane anion transporters: shedding light on anionophoric activity in cells. Chem. Sci. 2016, 7, 5069-5077. (46) Hill, A. V. The Combinations of Haemoglobin with Oxygen and with Carbon Monoxide. I. Biochem. J. 1913, 7, 471-480. (47) Clarke, H. J.; Van Rossom, W.; Horton, P. N.; Light, M. E.; Gale, P. A. Anion transport and binding properties of N Nʹ-(phenylmethylene)dibenzamide based receptors. Supramol. Chem. 2016, 28, 10-17. (48) Haynes, C. J. E.; Busschaert, N.; Kirby, I. L.; Herniman, J.; Light, M. E.; Wells, N. J.; Marques, I.; Felix, V.; Gale, P. A. Acylthioureas as anion transporters: the effect of intramolecular hydrogen bonding. Org. Biomol. Chem. 2014, 12, 62-72. (49) Spooner, M. J.; Gale, P. A. Anion transport across varying lipid membranes - the effect of lipophilicity. Chem. Commun. 2015, 51, 4883-4886. (50) Hussain, S.; Brotherhood, P. R.; Judd, L. W.; Davis, A. P. Diaxial Diureido Decalins as Compact, Efficient, and Tunable Anion Transporters. J. Am. Chem. Soc. 2011, 133, 1614-1617. (51) Lisbjerg, M.; Valkenier, H.; Jessen, B. M.; Al-Kerdi, H.; Davis, A. P.; Pittelkow, M. Biotin[6]uril Esters: Chloride-Selective Transmembrane Anion Carriers Employing C—H···Anion Interactions. J. Am. Chem. Soc. 2015, 137, 4948-4951. (52) Dias, C. M.; Li, H.; Valkenier, H.; Karagiannidis, L. E.; Gale, P. A.; Sheppard, D. N.; Davis, A. P. Anion transport by ortho-phenylene bis-ureas across cell and vesicle membranes. Org. Biomol. Chem. 2018, 16, 1083-1087. (53) Sakai, N.; Matile, S. The determination of the ion selectivity of synthetic ion channels and pores in vesicles. J. Phys. Org. Chem. 2006, 19, 452-460. (54) Jentzsch, A. V.; Emery, D.; Mareda, J.; Nayak, S. K.; Metrangolo, P.; Resnati, G.; Sakai, N.; Matile, S. Transmembrane anion transport mediated by halogen-bond donors. Nat. Commun. 2012, 3, 905. (55) Macchione, M.; Tsemperouli, M.; Goujon, A.; Mallia, A. R.; Sakai, N.; Sugihara, K.; Matile, S. Mechanosensitive Oligodithienothiophenes: Transmembrane Anion Transport Along Chalcogen-Bonding Cascades. Helv. Chim. Acta 2018, 101, e1800014. (56) Roy, A.; Saha, D.; Mukherjee, A.; Talukdar, P. One-Pot Synthesis and Transmembrane Chloride Transport Properties of C3-Symmetric Benzoxazine Urea. Org. Lett. 2016, 18, 5864-5867. (57) Roy, A.; Biswas, O.; Talukdar, P. Bis(sulfonamide) transmembrane carriers allow pH-gated inversion of ion selectivity. Chem. Commun. 2017, 53, 3122-3125. (58) Roy, A.; Gautam, A.; Malla, J. A.; Sarkar, S.; Mukherjee, A.; Talukdar, P. Self-assembly of small-molecule fumaramides allows transmembrane chloride channel formation. Chem. Commun. 2018, 54, 2024-2027.
(59) Avanti Polar Lipids Inc. https://avantilipids.com/product/840051 (accessed 09/08/2018 2018). (60) Avanti Polar Lipids Inc. https://avantilipids.com/product/850457 (accessed 10/08/2018 2018). (61) Wu, X.; Gale, P. A. Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters. J. Am. Chem. Soc. 2016, 138, 16508-16514. (62) Fischer, R. B. Ion-selective electrodes. J. Chem. Educ. 1974, 51, 387. (63) De Marco, R.; Clarke, G.; Pejcic, B. Ion-Selective Electrode Potentiometry in Environmental Analysis. Electroanalysis 2007, 19, 1987-2001. (64) Covington, A. K.: Ion Selective Electrode Method; CRC Press: Boca Raton, 1979. (65) Thermo Fisher Scientific Inc. http://www.thermofishersci.in/lit/Fisherbrand%20Accumet%20Electrodes%20Handbook.pdf (accessed 23/11/2018 2018). (66) Thermo Fisher Scientific Inc. https://www.fishersci.com/shop/products/thermo-scientific-orion-chloride-combination-electrode-mercury-free-chloride-combination/13620627 (accessed 28/11/2018 2018). (67) Thermo Fisher Scientific Inc. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/D12671~.pdf (accessed 22/11/2018 2018). (68) Mettler Toledo. https://www.mt.com/dam/non-indexed/po/ana/titration/ApplGuide_ISE_Cl_30253765_V04.15.pdf (accessed 23/11/2018 2018). (69) Thermo Fisher Scientific Inc. https://www.fishersci.co.uk/shop/products/orion-star-a211-ph-benchtop-meter/p-4529651 (accessed 27/11/2018 2018). (70) Avanti Polar Lipids Inc. https://avantilipids.com/divisions/equipment-products/mini-extruder-assembly-instructions/ (accessed 17/08/2018 2018).
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