A small synthetic molecule functions as a chloride-bicarbonate dual-transporter and induces chloride secretion in cells

Article abstract of DOI:10.1039/c6cc01964a

A C₂ symmetric small molecule composed of l-phenylalanine and isophthalamide was found to function as a Cl^-/HCO₃^- dual transporter and self-assemble into chloride channels. In Ussing-chamber based short-circuit current measurements, this molecule elicited chloride-dependent short-circuit current (I_sc) increase in both Calu-3 cell and CFBE41o-cell (with F508del mutant CFTR) monolayers.

Full text of DOI:10.1039/c6cc01964a

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A small synthetic molecule functions as a
chloride–bicarbonate dual-transporter and
Cite this:DOI: 10.1039/c6cc01964a
induces chloride secretion in cells†
Received 5th March 2016,
Accepted 3rd May 2016
Peng-Yun Liu,a Shing-To Li,a Fang-Fang Shen,a Wing-Hung Ko,*b Xiao-Qiang Yao*b
and Dan Yang*a
DOI: 10.1039/c6cc01964a
www.rsc.org/chemcomm
A C2 symmetric small molecule composed of L-phenylalanine and
isophthalamide was found to function as a ClÀ/HCO3 dual trans-
À
porter and self-assemble into chloride channels. In Ussing-chamber
based short-circuit current measurements, this molecule elicited
chloride-dependent short-circuit current (Isc) increase in both Calu-3
cell and CFBE41o-cell (with F508del mutant CFTR) monolayers.
Bicarbonate (HCO3À) plays important roles in biological pro-
cesses including photosynthesis, the Krebs cycle, whole-body
and cellular pH regulation, and volume regulation.1 In mammals,
movement of bicarbonate across the cell membrane is facilitated
by fourteen bicarbonate transporters, including the anion
exchanger (AE) family, Na+/HCO3 co-transport (NBC) family,
À
and Na-driven ClÀ/HCO3 exchanger (NDCBE), which drive
À
HCO3 metabolism.2 Dysregulation of bicarbonate transport
À
can lead to conditions such as cystic fibrosis,3 heart disease and
infertility.4 Previously, we discovered that a small C2-symmetric
molecule with an isophthalamide scaffold linking two R-(a)-
aminoxy leucine units (CM1) was capable of self-assembling
into selective ClÀ channels,5 and the isophthalamide scaffold
Scheme 1 Chemical structures of channel forming molecules.
linking two Boc-protected (a)-aminoxy lysine residues (CM2) can in liposomes. Compared with our previous studies, CM3 possesses
self-assemble into K+ channels in liposomes and living cell a simplified synthetic route and mucÀh higher transport abilities.
membranes (Scheme 1).6 As a-aminoxy acids have different While most studies on ClÀ/HCO3 transporters have been
conformational preference from regular a-amino acids, it will focused on their anticancer properties,7 we demonstrate here
be interesting to explore if the replacement of the a-aminoxy that CM3 mediates chloride and bicarbonate transport across
acid residues in both CM1 and CM2 by the corresponding Calu-3 monolayers and restores dysfunctional chloride secretion
a-amino acid residues would affect their ion transport activities. across CFBE41o-monolayers.
Herein, we report small molecule CM3, which has an isophthal-
Firstly, we used the pH-sensitive fluorescent dye 8-hydroxy-
amide scaffold linking two a-phenylalanine residues, can form 1,3,6-pyrene-trisulfonate (HPTS, pyranine) to test the ion trans-
chloride channels and mediate chloride/bicarbonate transport port properties of compounds CM3 and CM4 (an a-leucine
analog of CM3). HPTS and NaCl encapsulated liposomes were
a Morningside Laboratory for Chemical Biology, Department of Chemistry,
The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China.
E-mail: yangdan@hku.hk
b School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T.,
Hong Kong, P. R. China. E-mail: whko@cuhk.edu.hk, yao2068@cuhk.edu.hk
† Electronic supplementary information (ESI) available: Experimental procedures,
characterization data, and NMR spectra of new compounds (PDF). See DOI:
10.1039/c6cc01964a
suspended in an isotonic NaCl solution. Then a testing compound
and a NaOH base pulse were added to the extravesicular solution.
The resulting pH gradient caused by the base pulse drove the
efflux of protons or influx of hydroxide ions and built up a
transmembrane electrostatic potential. This electrostatic potential
could be compensated, if the testing compound mediated
anion efflux or cation influx and a subsequent continuous
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Fig. 3 SPQ assay on ClÀ/HCO3À exchange across liposome lipid bilayers.
Inside vesicles: 100 mM NaHCO3, 0.5 mM SPQ, pH 9.0; outside vesicles:
100 mM NaCl, 5 mM Tris, pH 9.0. At t = 50 s, THF (20 mL, negative control)
or a THF solution of CM1, CM3 or CM4 (20 mL, 20 mM final concentration)
was added.
Fig. 1 HPTS base pulse assay of channel forming molecules. Inside
vesicles: 100 mM NaCl, 0.1 mM HPTS, 10 mM HEPES, pH 6.8. Outside vesicles:
100 mM NaCl, 10 mM HEPES, pH 6.8. At t = 100 s, DMSO (20 mL, negative
control) or DMSO solution of testing compound (20 mL, 10 mM final concen-
tration) was added, followed by the addition of a NaOH aqueous solution (20 mL,
0.5 M). At t = 500 s, 40 mL of 5% Triton X-100 was added to lyse the liposomes.
solution at pH 9.0. In contrast to CM1, both CM3 and CM4
induced a decrease in the fluorescence of SPQ, indicating
HPTS fluorescence change would be recorded. As shown in
Fig. 1, by replacing the a-aminoxy acid residue of CM1 with the
a-amino acid residue, CM4 surprisingly mediated a more
efficient electrolyte exchange. Further side-chain modification
from leucine to phenylalanine enables CM3 to induce a more
rapid increase in HPTS fluorescence than CM4.
ClÀ/HCO3 exchange mediated by CM3 and CM4, but not by
À
CM1 (Fig. 3).
Since CM3 exhibited higher transport activity than CM4 in
both ClÀ transport and ClÀ/HCO3À exchange in liposome assays,
CM3 was chosen for further characterization experiments. The
patch-clamp study involving single-channel recording was
the most critical test for the formation of ion channels.9 The
characteristic single-channel current was recorded in the presence
of 10 mM CM3 in a bath solution of symmetric 150 mM NaCl
(Fig. 4) or symmetric 150 mM N-methylglucamine hydrochloride
(NMDG-Cl; Fig. S2, ESI†), indicating the formation of functional
chloride ion channels within the lipid bilayers of liposomes. In
addition, the relative ion permeability of ClÀ over Na+ was
determined to be 7.4 Æ 2.1 (Fig. S3, ESI†). Thus, our patch
clamp results were consistent with that obtained in SPQ fluorescence
experiments, further confirming that CM3 indeed forms chloride
channels in lipid bilayers.
To explore the ionic origin of electrolyte exchange in the
HPTS assay, another HPTS assay in Na2SO4 solution was carried
out (Fig. S1, ESI†). No continuous HPTS fluorescence change
was recorded, suggesting no Na+ transport in this assay. We
then used the halide-sensitive fluorescent indicator, 6-methoxy-
N-(3-sulfopropyl) quinolinium (SPQ),8 to compare the chloride
transport properties of CM1, CM3 and CM4 (Fig. 2). NaNO3 and
SPQ encapsulated liposomes were suspended in an isotonic
NaCl solution.5 Interestingly, CM3 and CM4 induced a more
rapid decrease of the fluorescence of SPQ than CM1, indicating
more efficient ClÀ influx mediated by CM3 and CM4.
À
Next, we used the SPQ assay to investigate the ClÀ/HCO3
À
The direct evidence for transmembrane HCO3 transport
exchange properties of CM1, CM3 and CM4. Liposomes encap-
sulating NaHCO3 and SPQ were suspended in an isotonic NaCl
À
facilitated by CM3 was obtained by the liposome-based H13CO3
NMR experiment (Fig. 5), as reported by Davis and coworkers.10
In this experiment, NaCl-encapsulated liposomes were suspended
in a NaH13CO3 buffer. Then paramagnetic Mn2+ was added to
the extravesicular soluÀtion to broaden the 13C-NMR signal of
extravesicular H13CO3 to the baseline, which allowed the
Fig. 2 SPQ chloride transport assay. Inside vesicles: 200 mM NaNO3,
0.5 mM SPQ, 10 mM HEPES, pH 7.0. Outside vesicles: 200 mM NaCl,
10 mM HEPES, pH 7.0. At t = 50 s, THF (20 mL, negative control) or a THF
solution of CM1, CM3 or CM4 (20 mL, 10 mM final concentration) was added.
Fig. 4 Typical single channel currents of self-assembled channels
recorded in the presence of CM3 at 10 mM, when both intra- and
extravesicular solutions were symmetric 150 mM NaCl.
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Fig. 6 Accumulated data of Ussing-chamber based short-circuit current
measurements (n = 4). For (a) and (b), short-circuit current was measured
in Calu-3 monolayers with low and high basal current, respectively. Unless
otherwise indicated, Calu-3 monolayers were bathed bilaterally with the
Krebs-Henseleit solution. Each column represents the mean Æ S.D. (n = 4).
***p o 0.001, **p o 0.005, *p o 0.01, quantitative differences between
extract groups values were statistically analyzed using one-way ANOVA
with a multiple comparison post hoc test by the Bonferroni method.
Fig. 5 13C-NMR spectroscopy experiments demonstrating that CM3
facilitated bicarbonate influx into vesicles. NaCl-encapsulated liposomes
were suspended in
a Na2SO4 solution. The intravesicular solution
contained 450 mM NaCl and 20 mM HEPES (pH 7.3), and the extravesicular
solution contained 150 mM Na2SO4 and 20 mM HEPES (pH 7.3). (i) The
13C-NMR spectrum was obtained after the addition of NaH13CO3 to the
extravesicular solution. (ii) The 13C-NMR spectrum was obtained after the
addition of 0.5 mM Mn2+(1/100 = [Mn2+]/[H13CO3À]). (iii-a) The 13C-NMR
spectrum was obtained after the addition of DMSO to the solution
obtained in step ii. (iii-b) The 13C-NMR spectrum was obtained after the
addition of CM3 to the solution obtained in step ii (CM3 in DMSO,
1% relative to lipid, the same volume as the DMSO added in step iii-a).
carbonate anhydrase (responsible for bicarbonate synthesis in
cells). It was found that the short-circuit current increase
elicited by CM3 was CFTR-independent, strongly chloride-
dependent and influenced by the presence of extracellular
bicarbonate ions or intracellular bicarbonate ion synthesis in
both batches of Calu-3 monolayers (Fig. 6a and b; Fig. S6 and S7,
ESI†).
In CFBE41o-cell monolayers with homozygous F508 mutation,
CM3 also induced chloride-dependent short-circuit current
increase (Fig. 7). After pretreatment with anion exchange inhibitor
DIDS or epithelial sodium channel (ENaC) inhibitor amiloride, the
short-circuit current response of CM3 was not greatly affected
(Fig. S8, ESI†). However, when the chloride gradient was
removed, the short-circuit current increase was almost abolished
(Fig. S8, ESI†), indicating the contribution of chloride to the
short-circuit current response. Therefore, we concluded that
CM3 restored the dysfunctional chloride secretion caused by
F508del mutant CFTR in the CFBE41o-cell line.
discrimination of the extravesicular and the intravesicular
HCO3À.ÀAfter the addition of CM3, the 13C-NMR signal of
H13CO3 was recovered, indicating an influx of the extra-
vesicular H13CO3À. As a negative control, the addition of DMSO
did not restore the H13CO3À NMR signal, indicating no influx of
the extravesicular H13CO3
.
À
Because the amide NH units of a-aminoxy acid residues are
more acidic than regular amide NH groups, they are expected
to be better hydrogen bond donors to interact with anions.
a-Aminoxy acid-containing compound CM1 indeed has higher
acidity (pKa 8.3, Fig. S4, ESI†) and ClÀ binding affinity (K 4 1 Â
105 MÀ1) than a-amino acid-containing compounds CM3 and
CM4 (with pKa 4 10). However, the ClÀ transport activities of
CM3 and CM4 were found to be superior to that of CM1. This
result suggests that the ClÀ binding affinity does not correlate
with the ClÀ transport activity. Instead, the ability of channel
assembly from small molecules in a non-polar lipid environment
may be more important than the ion binding affinity.
In summary, CM3 has been found to self-assemble to form
chloride channels in lipid bilayers and mediated ClÀ/HCO3
À
To explore the potential biomedical application of CM3, we
investigated the Ussing-chamber based short-circuit current
response of CM3 in Calu-3 and CFBE41o-epithelial monolayers,
which express functional CFTR and F508del-mutated CFTR,
respectively.11 Two batches of Calu-3 monolayers were used,
one with low basal current (6.4 Æ 0.8 mA cmÀ2, n = 30) and the
other one with high basal current (19.82 Æ 1.28 mA cmÀ2
,
n = 32). In both monolayers, CM3 elicited short-circuit current
increase (Fig. 6a and b; Fig. S6 and S7, ESI†). Calu-3 monolayers Fig. 7 Accumulated data of Ussing-chamber short-circuit current
measurement of CM3 in CFBE41o-monolayers (n = 4). Without special
notification, CFBE41o-monolayers were bathed with basolateral to
were then pretreated with different channel inhibitors (Fig. S6,
ESI†) to explore the ionic nature of short-circuit current increase:
CFTRinh-172 inhibited apical CFTR conductance; bumetanide
blocked ClÀ uptake through a sodium potassium chloride
apical chloride gradient. Each column represents the mean
(n = 4). ***p o 0.001, quantitative differences between group values were
statistically analyzed using one-way ANOVA with a multiple comparison
Æ
S.D.
cotransporter (NKCC); acetazolamide suppressed intracellular post hoc test by the Bonferroni method.
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7 N. Busschaert and P. A. Gale, Angew. Chem., 2013, 52, 1374;
transport across liposome membranes. In Ussing-chamber
based short-circuit current measurements, CM3 has been
shown to elicit short-circuit current increase in Calu-3 epithelial
monolayers, which could be attributed to the secretion of
chloride, and influenced by bicarbonate ions. It can also restore
the chloride secretion in CFBE41o-monolayers with F508del
mutant CFTR. This discovery opens up an opportunity for the
treatment of channelopathies caused by dysfunction of chloride
and bicarbonate channels, notably cystic fibrosis.
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