Controlling epithelial sodium channels with light using photoswitchable amilorides

Article abstract of DOI:10.1038/nchem.2004

Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane proteins, assembled from β, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx into non-epithelial cells. The functional role of δβγENaC in various organs, including the human brain, is still poorly understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical control of ENaC channels, in particular the δβγ isoform, by switching between blue and green light, or by turning on and off blue light. PA1 was used to modify functionally δβγENaC in amphibian and mammalian cells. We also show that PA1 can be used to differentiate between δβγENaC and αβγENaC in a model for the human lung epithelium.

Full text of DOI:10.1038/nchem.2004

ARTICLES
PUBLISHED ONLINE: 20 JULY 2014 | DOI: 10.1038/NCHEM.2004
Controlling epithelial sodium channels with light
using photoswitchable amilorides
Matthias Schönberger1, Mike Althaus2, Martin Fronius2,3, Wolfgang Clauss2 and Dirk Trauner1
*
Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane
proteins, assembled from β, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx
into non-epithelial cells. The functional role of δβγENaC in various organs, including the human brain, is still poorly
understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing
ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical
control of ENaC channels, in particular the δβγ isoform, by switching between blue and green light, or by turning on and
off blue light. PA1 was used to modify functionally δβγENaC in amphibian and mammalian cells. We also show that PA1
can be used to differentiate between δβγENaC and αβγENaC in a model for the human lung epithelium.
he epithelial sodium channel (ENaC) is a constitutively open helices (TM1 and TM2) with relatively short intracellular C and
sodium-selective ion channel that lacks voltage sensitivity N termini and a large, cysteine-rich extracellular domain.
T
and mediates sodium absorption across various epithelia1.
Although ENaC/deg channels respond to a variety of input
Classical ENaCs are heterotrimers composed of three subunits, α, signals, none are inherently sensitive towards light. In recent
β and γ, which assemble as a sodium-selective ion channel in the years, our group succeeded in developing a range of synthetic
plasma membrane (Fig. 1)2,3. In renal epithelia, ENaCs mediate photoswitches that can confer light sensitivity to native or slightly
Na+ reabsorption by the kidney and are involved in maintaining modified receptor proteins. These can either function as freely dif-
extracellular volume and blood pressure. Gain-of-function fusible photochromic ligands (PCLs) or as photoswitchable tethered
mutations cause a hereditary form of hypertension (Liddle syn- ligands, which are covalently attached to the target protein25. Using
drome), whereas loss-of-function mutations give rise to the salt- either strategy, effectively we were able to turn a variety of ion
wasting syndrome pseudohypoaldosteronism type 14,5. ENaC- channels and G-protein-coupled receptors into photoreceptors26–30
mediated sodium absorption by the pulmonary epithelium drives We now show that an entirely new class of transmembrane pro-
.
lung liquid clearance and is thus a critical regulator of pulmonary teins, namely trimeric ENaC/deg channels, are amenable to the PCL
water content. Increased ENaC activity in the airways is the approach. To this end we have designed, synthesized and character-
cause of cystic-fibrosis-like lung disease, whereas decreased ENaC ized a small series of photoswitchable azobenzene derivatives of
activity in the distal lung leads to impaired alveolar fluid clearance amiloride and identified one candidate that can be used primarily
and the development of pulmonary oedema6–9. As such, ENaCs to control δβγENaC activity with light. Our photochromic ligands
are a primary target for drugs that affect electrolyte and water provide insights into the structure–activity relationships of ENaC
homeostasis, such as diuretics or oedema medications10–13
.
blockers and could be used to discriminate functionally
An additional ENaC-subunit, δ, was identified in 199514. There is between isoforms.
only scarce knowledge about its physiological function; however, it
can assemble with the β and γ subunits to form channels with a high Results and discussion
open probability14–16. Expression of δENaC (and other ENaCs) has Design and synthesis of photoamilorides. Amiloride was
been confirmed in various non-epithelial tissues, most prominently introduced as a potassium-sparing diuretic in the 1960s without
the central nervous system of primates17–20. It has been speculated particular knowledge of its molecular target31. Subsequently,
that δ-containing ENaCs play a role in communicating ischaemic ENaC in kidney epithelia was established as the ion channel
and hypoxic signals in neurons, as well as in adjusting nervous excit- primarily responsible for the potassium-sparing diuretic effect3.
ability21,22. Complicating the analysis, rodents do not appear to have To date, countless amiloride analogues have been synthesized and
a gene for the δ subunit. Furthermore, no pharmacological tools are tested, which has provided insights into its structure–activity
available for functional differentiation between α- and δ-containing relationships32,33. Most importantly, it has been found that
ENaCs. Thus, in contrast to αβγENaC, little is known about the lipophilic substitution at the terminal guanidine nitrogen boosts
physiological function of δβγENaC and, in particular, the function both potency and selectivity for ENaC. Indeed, the potency
of δENaC in the brain22.
gained by an aromatic substituent increases with linker length
ENaCs belong to a large group of ion channels, the ENaC/degen- (Fig. 1b)34. Accordingly, we designed photochromic amiloride
erin (ENaC/deg) protein family, which also includes neuronal acid- derivatives that have an azobenzene unit attached directly to
sensing ion channels23. Recently, the latter were elucidated with the guanidine, which gave photoamiloride-1 (PA1) and
X-ray crystallography, which confirmed a trimeric architecture photoamiloride-2 (PA2), both of which resemble the drug
that probably applies to all channels of the family (Fig. 1)24. Every phenamil (Fig. 1). Phenamil is a long-lasting blocker of ENaCs
individual ENaC subunit comprises only two transmembrane and has been used to determine channel density35. In addition, we
1Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians-Universität München, Butenandtstraße 5–13 (F4.086), 81377
Munich, Germany, 2Institute of Animal Physiology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany, 3Department of
Physiology, University of Otago, PO Box 913, Dunedin 9054, New Zealand. e-mail: dirk.trauner@lmu.de
*
712
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
a
c
b
Na+
N
NH2
O
NH
NH2
O
NH2
N
N
N
H
NH2
N
N
H
N
γ
α or δ
β
N
N
H2N
H2N
PA1
Amiloride
N
Cl
Cl
N
NH2
O
NH2
NH2
O
NH2
N
N
N
H
N
N
N
H
N
N
N
H2N
H2N
H2N
H2N
PA2
Phenamil
Cl
Cl
N
NH2
O
NH
NH2
O
NH2
N
N
N
H
N
H
N
N
H
N
ex
N
N
H2N
PA3
Benzamil
N
Cl
Cl
N
NH2
O
NH
NH2
O
NH2
N
in
N
N
H
N
H
N
N
H
N
C
N
N
H2N
N
1
PA4
Cl
Cl
N
d
Amiloride
Azobenzene
N
N
NH2
O
NH
NH2
O
NH
N
400 nm
500 nm
N
N
H
N
H
N
N
H
N
H
N
N
H2N
H2N
Cl
Cl
trans-PA1
cis-PA1
Figure 1 | ENaCs and their blockers. a, Schematic drawing of heterotrimeric αβγENaC and δβγENaC. Each subunit contains two transmembrane helices TM1
and TM2, relatively short C and N termini at the intracellular (in) site and a large extracellular (ex) loop. Under physiological conditions, ENaCs allow for a
constant influx of sodium into the cell (indicated by blue arrows). b, Chemical structure of amiloride and its more potent derivatives phenamil, benzamil and
compound 1. c, Structures of photoswitchable amiloride derivatives that contain an azobenzene functional group. d, Photoisomerization of PA1. Illumination
with 400 nm induces isomerization to cis-PA1, which is the thermodynamically less-stable form. The cis isomer can relax back to the trans state thermally, or,
more rapidly, by illumination using 500 nm radiation.
designed the compounds photoamiloride-3 (PA3) and coupling conditions as PA1 and PA2 (and vice versa) failed.
photoamiloride-4 (PA4), which bear an azobenzene moiety linked Similarly, attempts to condense 4-aminoazobenzenes 2 and 3 with
through an ethylene spacer and resemble the known amiloride carboxamidine 8 did not yield the corresponding guanidines.
analogue 136. In PA2 and PA4, the azobenzenes are substituted
A single-crystal X-ray structure of PA1 is shown in Fig. 2c. It pro-
further in the 4′-position with a diethylamino substituent, which vides the first structural data of an amiloride derivative substituted at
is known to red shift the action spectra of azobenzenes37. The the terminal guanidine nitrogen and reveals an extended hydrogen-
photoswitching of PA1 between its trans and cis configuration is bonding network that involves an amine on the pyrazine, the
shown in Fig. 1d.
adjacent carbonyl and the guanidine (dotted lines).
The synthesis of the shorter photoamilorides PA1 and PA2
started with aminoazobenzenes 2 and 3, respectively, which were Photophysical characterization of photoamilorides. To determine
converted into aryl guanidines using cyanamide (Fig. 2a and absorption maxima and the optimal wavelengths for
Supplementary Information). The resulting guanidine hydrochlo- photoswitching, solutions of PA1-4 (50 µM, dissolved in
rides were deprotonated and condensed with an active ester of dimethylsulfoxide (DMSO) were irradiated at different
pyrazinic acid 4, which yielded PA1 and PA2, respectively.
wavelengths using a monochromator (Till Photonics Polychrome
To generate the longer photoamilorides, aniline 5 was condensed 5000, Supplementary Fig. 1). DMSO was chosen as the solvent to
with nitrosobenzene and subsequently deprotected to yield 6 slow down the thermal relaxation to resolve the absorption
(Fig. 2b and Supplementary Information). Alternatively, oxidation spectra of cis and trans isomers for red-shifted candidates38. Thus,
of 5 and condensation with p-(diethylamino)aniline, followed by it was possible to identify wavelengths that led to the highest cis
deprotection, gave azobenzene 7. With these two building blocks and trans content for each molecule (Fig. 2d).
in hand, the guanidine moiety was installed using carboxamidine
8. The resulting guanidinium salts were deprotonated via a basic Electrophysiological characterization of photoamilorides in the
work-up and added directly to an ethanolic solution of ester 9. dark. To identify the amiloride derivative that would be the
Heating to reflux then yielded PA3 and PA4, respectively. best photochromic ENaC blocker, initially we performed
Interestingly, attempts to furnish PA3 or PA4 under the same microelectrode recordings on Xenopus oocytes that express
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713

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
a
i
H2NCN, HCl
(51%, 65%)
N
R
NH2
O
NH2
N
PA1, R = H (57%)
N
N
N
N
H
N
PA2, R = NEt2 (73%)
H2N
N
NH2
N
H2N
ii
H2N
O
Cl
N
Cl
N
OH
4
NH
N
b
NH2
NH2
O
NH2
N
NHCl
N
C6H5NO
then TFA
8
N
N
N
H
N
N
N
H2N
(80%)
NHBoc
6
H2N
N
NH2
Cl
PA3 (38%)
then
5
NH2
O
Cl
N
OMe
9
Oxone
NEt2
then H2NC6H4NEt2
then TFA
NEt2
N
NH2
O
NH2
N
(49%)
N
N
8
N
N
H
N
7
N
then 9
H2N
PA4 (15%)
NH2
Cl
c
d
e
Switching wavelengths (nm)
IC50 values (nM)
αβγ
90
δβγ
390
8,000
80
δ/α
4.3
λ (cis)
400
λ (trans)
500
PA1
PA1
PA2
PA3
PA4
PA2
PA3
PA4
346
36
23.1
2.2
440
600
360
500
PA1
4
14
3.5
420
560
Figure 2 | PA synthesis and characterization. a, Synthesis of PA1 and PA2 starts with 4-aminoazobenzenes 2 and 3, respectively. Reaction with cyanamide
installs guanidine functional groups, which can be coupled with an active ester of 4 to yield PA1 and PA2. b, Synthesis of PA3 and PA4 starts from the
protected phenethylamine 5, which can be either condensed with nitrosobenzene or oxidized to a nitrosobenzene and condensed with p-(diethylamino)
aniline. After acidic deprotection, primary amine building blocks 6 and 7 are functionalized to guanidines, which can react with ester 9 to yield PA3 and PA4,
respectively. c, X-ray structure of PA1 indicates an extended hydrogen-bond network (dotted lines). d, Optimal wavelengths for photoswitching were
determined using UV/vis spectroscopy. Generally, the more conjugated candidates PA1 and PA2 are more red-shifted than their corresponding homologues
PA3 and PA4. Substitution with the electron-donating diethylaniline functionality leads to a red shift compared to the unsubstituted candidates (PA2 and
PA4 versus PA1 and PA3). e, IC50 values of PAs in their dark-adapted trans form. PA4 is the most-potent and PA2 is the least-potent ENaC blocker. All PA
molecules are more potent on the αβγ isoform. PA2 discriminates the most between αβγENaC and δβγENaC.
human αβγENaC and δβγENaCs for all the molecules of a C-2 linker markedly increased affinity, as PA3 and PA4 are sub-
(Supplementary Fig. 2). In the presence of a dim room light to stantially more potent than their shorter analogues. The addition of
ensure that PAs were in their trans form, a series of increasing a substituent in the 4′-position in PA4 further increased potency
ligand concentrations was applied, followed by full block using a compared to the unsubstituted PA3. However, this trend is reversed
100 µM amiloride solution. This enabled both the determination when there is no linker between the guanidine and the
of half-maximum inhibitory concentration (IC50) values and a azobenzene moiety.
relative block compared to the full blocker amiloride (Fig. 2e and
Supplementary Fig. 2). It was found that all the photochromic Photocurrents in Xenopus oocytes. To investigate which
ligands synthesized were ENaC blockers. In general, they have a compound elicits the most-pronounced photoeffect, we performed
higher affinity for α-containing than δ-containing ENaCs. PA4 is electrophysiological recordings on Xenopus oocytes that
the most potent molecule with IC50 values of 4 and 14 nM for expressed either αβγENaC or δβγENaC in combination with
αβγENaC and δβγENaC, respectively. The weakest blocker is PA2 monochromatic illumination. PA1–PA4 were applied at varying
with IC50 values of 346 and 8,000 nM for αβγENaC and concentrations and the illumination wavelengths were switched in
δβγENaC, respectively. For comparison, amiloride has IC50 values intervals of one minute as the perfusion was stopped
of 100 nM and 2,600 nM on αβγENaC and δβγENaC channels, (Supplementary Fig. 3). Among our four amiloride derivatives,
respectively, in a similar experimental setting.22
PA1 emerged as the most-efficient photochromic blocker,
The structure–activity relationships of our small molecular series especially when applied to the δβγ isoform. Our further
provided additional interesting findings (Fig. 2e). Matching the investigations therefore focused on this compound. As shown in
pharmacology of previously known ENaC blockers, the addition Fig. 3, illumination of PA1 with 400 nm radiation immediately
714
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
a
c
PA1 (10 μM)
αβγENaC δβγENaC
b
δβγENaC, 10 μM PA1
δβγENaC, 10 μM PA1
0.0
–0.5
–1.0
–1.5
0.8
0.6
0.4
0.2
400 420 440 460 400 nm
*
*
10 s
5 s
0.0
500 nm
400 nm
500 nm
500 nm
400 nm
αβγ δβγ
1 min
10 s
Figure 3 | PA1 photoswitching in Xenopus oocytes. a, Reversible block of δβγENaC currents evoked by photoisomerization of PA1 (10 µM) in 10 s
and 5 s intervals. b, Action spectrum of PA1. Wavelengths were switched between 500 nm and 400 + x nm (x = 0, 20, 40, 60). The largest photoswitchable
currents are observed when 500 and 400 nm are used as illumination wavelengths for cis and trans isomerization. c, Photoswitching of PA1 tested on an
amiloride-sensitive current (IA) of αβγENaC-expressing (left) and δβγENaC-expressing (right) Xenopus oocytes by two-electrode voltage clamp recordings.
Perfusion was stopped and illumination wavelengths were switched between 500 and 400 nm in one minute intervals. Current values were taken at the end
of each interval and averaged (n = 12 and 10, respectively). Error bars indicate s.e.m., and significance refers to switching from 400 nm to 500 nm (*p <
0.05). There is a reversible photoswitching effect on both αβγENaC and δβγENaC. However, the amplitude of PA1-evoked photoswitching is higher on
δβγENaC.
triggered a strong reduction in current. This effect could be reversed δβγENaC translates into a pronounced and fast light-dependent
by switching back to 500 nm (Fig. 3 and Supplementary Fig. 4a). change in membrane potential. Photoeffects are generally higher
Thus, PA1 functions as a cis blocker of δβγENaC. We found that on the δβγ isoform over the tested range of 10 nM to 25 µM PA1
the overall switching amplitude evoked by PA1 is consistent and the difference between αβγENaC and δβγENaC increases
between 1 and 10 µM and decided to settle on 10 µM as a with higher concentrations (Supplementary Fig. 6c,d).
A
working concentration, which provides maximal block in the comparison of photoswitching using HEK cells expressing either
trans state, close to a full amiloride block (Supplementary δβγENaC or αβγENaC and 10 µM PA1 revealed that the average
Fig. 4b). Control experiments using oocytes that expressed ENaC amplitude of the photocurrents is ∼180 pA on the former and
channels showed no innate sensitivity to light (Supplementary ∼10 pA on the latter isoform. Similarly, the average change in
Fig. 5). Similarly, ENaC-expressing oocytes blocked by a 100 µM membrane potential was ∼57 mV and ∼4 mV, respectively
solution of amiloride did not respond to illumination39.
(Fig. 4b; overall expression was identical (Supplementary Fig. 8)).
Figure 3a shows the effect of PA1 on a δβγENaC-mediated Thus, the general trends and effects that were found in Xenopus
current. Wavelengths, and consequentially currents, were switched oocytes could also be observed in HEK cells, but much more
alternately in intervals of ten and five seconds. With light-mediated pronounced and with a faster onset (Supplementary Fig. 7). We
activation, perfusion-induced mechanical effects can be avoided, as also found that cis-PA1 thermally relaxes back to trans-PA1 on a
recently demonstrated for ENaCs as well as for other members of fast time scale (Fig. 4c). Thus, our novel optical tool can be
the ENaC/deg family40–43
.
operated by turning on and off a simple light source. As in the
The photostationary state of azobenzenes, that is their cis/trans case of oocytes, intermediate wavelengths between 400 and
ratio, is a function of the illumination wavelength, which allows 500 nm translate into graded effects (Fig. 4d). We were thus able
for controlling their effective concentrations with light. As a conse- to induce current and voltage steps in δβγENaC-expressing HEK
quence, the degree to which δβγENaC channels are blocked by PA1 cells by switching between 400, 433, 466 and 500 nm.
also depends on the wavelength applied (Fig. 3b). Switching in
To determine further the potential of PA1 as a tool for adjusting
20 nm steps between a given wavelength and 500 nm results in a membrane potential, we set the starting potential in HEK cells in the
graded effect on the current. In line with our previous ultraviolet/ current–clamp mode at –70 mV, initially using either blue or green
visible (UV/vis) experiments, 400 nm radiation emerged as the illumination (Fig. 4e). Starting from these conditions, PA1 can be
most effective blocking wavelength. As shown in Fig. 3c, photo- used not only to hyperpolarize (switching from 500 to 400 nm),
switching mediated by 10 µM PA1 could be detected on both but also to depolarize (switching from 400 to 500 nm) a cell. As
αβγENaC- and δβγENaC-expressing oocytes. However, switching such, the δβγENaC–PA1 system could be used as an
events were generally more robust and amplitudes were higher on optogenetic tool.
δβγENaC (also see Supplementary Fig. 6).
Pharmacokinetics of PA1 in HEK cells. In the course of our
PA1 photoswitching in human embryonic kidney cells. We next experiments using PA1 on HEK cells expressing δβγENaC, we
turned to human embryonic kidney (HEK293t) cells as a found that photoswitching is persistent and channel block remains
mammalian expression system. Given that these cells are smaller after washout of PA1 from the external recoding solution
and transparent, we expected faster and more pronounced effects (Supplementary Fig. 9a,b). This interesting observation hints at
compared to those of the pigmented oocytes, for which only a a possible cell uptake or a reservoir formation in the cell
fraction of the membrane surface can be illuminated. As depicted membrane. Interestingly, Xenopus oocytes did not take up PA1 as
in Fig. 4, PA1 is an excellent photoswitchable blocker of readily as HEK cells, which might be another reason for the
δβγENaC-expressing HEK cells. In line with the data from difference in overall photoeffects (Supplementary Fig. 9c). To
Xenopus oocytes, 400 nm radiation translates into a rapid block of investigate this finding further, we preincubated coverslips with
ENaC currents, which can be released with 500 nm radiation HEK cells expressing δβγENaC for one minute at room
(Fig. 4a). In the current-clamp mode, the action of PA1 on temperature in 10 µM PA1. Coverslips were then washed three
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
a
c
b
d
αβγ
433 nm
δβγ
200
150
100
50
466nm
(10)
(10)
5 s
0
–30
60
–40
40
20
0
–50
–60
694
nm
(10)
400
500
nm
(10)
5 s
5 s
e
f
Patch–
clamp
r.t., 1 min
Hyperpolarization
–40
Wash buffer
10 μM PA1
–60
–70
–80
–60
–70
–80
Depolarization
–100
–120
3 s
10 s
Figure 4 | Switching δβγENaC in HEK cells using 10 μM PA1. a, By switching between 400 and 500 nm both large currents (upwards deflection =
inhibition) and potentials (downwards deflection = hyperpolarization) can be controlled by light. b, The average current amplitude of photoswitching (ΔIP)
is 182 pA for δβγENaC and 9 pA for αβγENaC. The average amplitude of the light-mediated membrane potential (ΔVP) is 57 mV for δβγENaC and 4 mV for
αβγENaC (currents and potentials were recorded from the same cell, n = 10 biological repeats from three independent transfections and holding potential was
–60 mV). c, Comparison between light-induced and thermal relaxation. d, Application of light with wavelengths between 400 nm and 500 nm reveals the
physiological action spectrum of PA1 on δβγENaC-expressing HEK cells. Graded effects can be installed for both current (top) and membrane potential
(bottom). e, Light-induced hyperpolarization and depolarization in the current-clamp mode. When the starting potential is adjusted to –70 mV under green
light (left), photoswitching leads to hyperpolarization. When the starting potential is adjusted to –70 mV under blue light (right), photoswitching leads to
depolarization. f, PA1 can be loaded into HEK cells by a short incubation. After one minute incubation at room temperature (r.t.) in 10 µM PA1 (yellow Petri
dish) and subsequent wash (blue Petri dish), cells are ready for photoexperiments using the patch–clamp technique.
times and investigated using whole-cell patch–clamp experiments. and δβγENaC in HEK cells, we next wanted to employ PA1 in a
We obtained a fully functional photoswitchable system with strong, mammalian tissue model that endogenously expresses ENaC
persistent ENaC photocurrents (Fig. 4f). Figure 4f further subunits. Human H441 bronchiolar epithelial cells were the
demonstrates the rapid robust and reversible action of PA1 on model of choice because these cells (1) express all four ENaC
δβγENaC. Intriguingly, cells prepared in such a manner could be subunits, including δENaC16, and (2) form a polarized epithelium,
blocked and unblocked by amiloride without affecting which allows the electrophysiological assessment of sodium
photoswitching (Supplementary Fig. 10). However, photoswitching transport across an intact cell monolayer. Furthermore, despite
was reduced when phenamil was used as a blocker with similar the molecular evidence for δENaC expression in pulmonary
lipophilicity, which is also taken up by cells (Supplementary epithelia,
Fig. 10c). In addition, we loaded PA1 through the patch pipette transepithelial sodium transport has not been demonstrated22.
(Supplementary Fig. 11). Photoswitching was possible To address the functional role of δβγENaC in these cells, we
a
functional contribution of this isoform to
instantaneously and a reversible amiloride block was also observed. measured the transepithelial potential of H441 monolayers,
Additional PA1 delivered by perfusion did not enhance the which were cultured on permeable membrane supports
photoeffect. These observations suggest that PA1 finds its binding (Fig. 5a). First, we determined the magnitude of the overall ami-
site from a membrane reservoir and has similar pharmacokinetics loride-sensitive transepithelial potential (VE) (on average ∼4 mV,
to long-lasting, use-dependent open channel blockers44.
Fig. 5c). We then exchanged the apical bath solution for 10 µM
PA1 and switched between 400 and 500 nm, and 400 nm and
Use of PA1 in a lung-tissue model. With the ability of PA1 to 694 nm for three cycles, respectively (Fig. 5b). The deep-red wave-
switch ENaC currents and to differentiate between αβγENaC length functionally corresponds to darkness because our
716
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
a
c
Voltmeter
b
5.0
4.5
4.0
3.5
Light
source
500 /400 nm
*
0.1
*
*
10 μM
V
0.0
–0.1
–0.2
–0.3
amiloride
*
*
*
10 μM
PA1
–0.4
PA1
–0.5
500/400
Dark/400
nm
–0.6
nm
0.6
0.5
0.4
0.3
0.2
0.1
0.0
30 s
H441 ML
–0.7
*
αβγ
δβγ
*
d
0.0
–0.1
–0.2
–0.3
–0.4
–0.5
–0.6
–0.7
0.1
0.0
+
694 /400 nm
*
Na
Li+
Na+ Li+
*
*
–0.1
–0.2
–0.3
–0.4
–0.5
–0.6
–0.7
*
*
*
n.s.
30 s
Li+
+
Li+
Na+
(7)
Na
*
Figure 5 | Photocontrol of transepithelial potential in H441 monolayers. a, Experimental configuration with an H441 monolayer (ML) on a filter inlet. The
apical compartment contained 10 µM PA1 in sodium- or lithium-based buffer and was illuminated with a monochromator. The transepithelial potential VE
between the apical and basolateral compartment was measured by a voltmeter (apical electrode is the reference). b, VE can be switched by toggling between
500 nm and 400 nm radiation (top), as well as between 694 nm and 400 nm (bottom). The 400 nm radiation was applied for ten seconds, the longer
wavelengths for 20 seconds and the voltage was recorded at the end of each illumination period. Photoeffects are robust and reversible for both protocols.
c, Comparison of the entire amiloride-sensitive VE and PA1-mediated photosensitive VE with apical sodium or lithium using both 400/500 nm and 400 nm/
dark switching (n = 7). d, Comparison of the effect of extracellular lithium on αβγENaC and δβγENaC photocurrents in Xenopus oocytes (n ≥ 7). Error bars
indicate s.e.m., *p-value < 0.05.
azobenzenes do not absorb in this region. Results were identical of ENaC channels. PA1 can be used to control δβγENaCs optically
for the different wavelength protocols and the overall magnitude by switching between blue and green light, or by turning on and off
was a photosensitive transepithelial potential VE of ∼0.4 mV blue light. This therefore allows ENaC to be investigated without
(Fig. 5b). This finding hints towards a small amount of functional mechanical stress. We showed that PA1 is long lasting and can be
δβγENaC. To investigate whether the photoeffect comes from applied by perfusion, cell incubation or loading through the patch
δβγENaC or is a small effect of αβγENaC, we addressed the pipette. We used PA1 to address the significance of different
lithium conductivity of each isoform. The ratio between lithium ENaC isoforms in H441 monolayers and identified functional
and sodium conductivity is 0.64 for δβγENaC and 2.0 for δβγENaC. The combination of photoswitchable PA1 and the
αβγENaC22. Thus, we expected decreased photoeffects for the voltage-insensitive δβγENaC is a powerful tool to control precisely
δ-containing isoform and increased effects for the α-containing the membrane potential with light, allowing for both hyper- and
isoform when sodium is replaced by lithium. We confirmed this depolarization. With its unique functional profile, PA1 could
hypothesis using Xenopus oocytes (Fig. 5d), which had shown enable new insights into the δβγENaC function, for instance in
photoeffects for either isoform before. Similarly, δβγENaC photo- the primate brain.
currents were significantly reduced in HEK cells by extracellular
Methods
lithium (Supplementary Fig. 12). When sodium was substituted
by lithium at the apical side of the H441 monolayer, PA1-
mediated photoeffects decreased from 0.4 to 0.15 mV for both
wavelength protocols (Fig. 5c). Thus, the photosensitive fraction
of VE results from δβγENaC rather than from αβγENaC. These
data strongly indicate a functional expression of δβγENaC in
H441 cells and underline the ability of PA1 for functional differ-
entiation between αβγENaC and δβγENaC.
The Supplementary Information gives details of chemical syntheses and Xenopus
oocytes cultures.
Cell culture and electrophysiology. HEK293t cells were cultured in DMEM
buffer with 10% fetal bovine serum in a 10% CO2 37 °C incubator. At 90–95%
confluency, cells were split and used for transient transfection using the jetPRIME
polyplus transfection reagent in accordance with the supplier’s protocol. Poly-L-
lysine-coated acid-etched glass coverslips were placed in a 24-well plate and
consecutively treated with the DNA mix (150 ng of each ENaC subunit and 50 ng
yellow fluorescent protein (YFP)) and 30,000 to 50,000 cells in growth medium.
After 2–4 hours, the supernatant was removed and growth medium supplemented
with 10 µM amiloride was added. Cells were used for electrophysiological
recordings 12–48 hours post-transfection. Only YFP-positive cells with an
amiloride-sensitive current were used for the experiments. For each repeat, a new
cover slip was used.
Conclusion
In summary, the photoswitchable ENaC blocker PA1 has been
designed, synthesized and functionally characterized using electro-
physiology with Xenopus oocytes, HEK293t cells and H441 cell
monolayers. Our results provide new insights into the structure–
activity relationships of the amiloride class of diuretics and
Whole-cell patch–clamp experiments were performed using a standard
electrophysiology set-up equipped with a HEKA Patch Clamp EPC10 USB amplifier
outline a strategy to address selectively the αβγ and δβγ isoforms and PATCHMASTER software. Micropipettes were generated from Science
NATURE CHEMISTRY | VOL 6 | AUGUST 2014 | www.nature.com/naturechemistry
717
© 2014 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
Products GB200-F-8P with filament pipettes using a vertical puller. Resistance
varied between 5 and 8 MΩ. The bath solution contained (in mM) 140 NaCl (or
LiCl), 3 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, 20 HEPES (NaOH to pH 7.4). The
pipette solution contained (in mM): 90 K gluconate, 10 NaCl, 10 KCl, 1 MgCl2, 10
EGTA, 60 HEPES (KOH to pH 7.3). Photoswitchable ligands and reference agonist
were dissolved in bath solution from a 1,000× DMSO stock.
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H441 cells were purchased from American Type Culture Collection in the
65th passage and cultured under liquid/air conditions in the presence of 200 nM
dexamethasone, as previously described45. For the experiments, H441-containing
cell culture inserts were placed in six-well plates containing H441 saline (2 ml in
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MgCl2, 10 HEPES, 10 glucose (37 °C, pH 7.35 (TRIZMA)). VE was measured
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exposed to different wavelengths. Afterwards, cells were rinsed with lithium-
containing saline and the same measurement protocol was employed.
Experiments were performed at 37 °C on seven H441 monolayers from two
independent cell preparations.
Irradiation was performed on a TILL Photonics Polychrome 5000
monochromator. In patch–clamp experiments, the light beam was guided
through the microscope objective and operated by the amplifier and
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Received 7 May 2013; accepted 12 June 2014;
published online 20 July 2014
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718
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© 2014 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2004
ARTICLES
(AL1453/1-1 and AL1453/1-2), M.F. and W.C. acknowledge a grant provided by the Federal
State of Hesse (LOEWE Research Focus, Non-neuronal cholinergic systems). M.S. was
supported by a grant from the German Study Foundation and the International Max Planck
Research School (IMPRS-LS). We acknowledge the support of K. Hüll (chemical synthesis)
and L. de la Osa de la Rosa (cell-culture work), and we thank D. Barber for helpful
comments. We also thank D. Alvarez de la Rosa for providing constructs of all ENaC
subunits in pcDNA3.1.
the electrophysiological characterization. D.T. and W.C. supervised the study and wrote the
paper, together with M.S., M.F. and M.A.
Additional information
Supplementary information and chemical compound information is available in the online
version of the paper. Reprints and permissions information is available online at www.nature.
com/reprints. Correspondence and requests for materials should be addressed to D.T.
Author contributions
D.T., M.A., M.S. and M.F. conceived the study and designed the experiments. M.S.
performed the chemical synthesis and UV/vis characterization. M.S. and M.A. performed
Competing financial interests
The authors declare no competing financial interests.
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