Chloride transport across lipid bilayers and transmembrane potential induction by an oligophenoxyacetamide

Article abstract of DOI:10.1021/ja029372t

This contribution describes the discovery and properties of a synthetic, low-molecular weight compound that transports Cl- across bilayer membranes. Such compounds have potential as therapeutics for cystic fibrosis and cancer. The H⁺/Cl^-

Full text of DOI:10.1021/ja029372t

Published on Web 02/13/2003
Chloride Transport Across Lipid Bilayers and Transmembrane Potential
Induction by an Oligophenoxyacetamide
Vladimir Sidorov, Frank W. Kotch, Jennifer L. Kuebler, Yiu-Fai Lam, and Jeffery T. Davis*
Department of Chemistry and Biochemistry, UniVersity of Maryland at College Park, College Park, MD, 20742
Received November 16, 2002 ; E-mail: jd140@umail.umd.edu
Identification of compounds that transport chloride across cell
membranes has important implications in drug development. A new
strategy for treating diseases caused by chloride channel malfunc-
tions, such as cystic fibrosis, relies on synthetic Cl- transporters.1
Peptides based on sequences thought to line the pore of natural
chloride channels can restore defective Cl- transport.2 Compounds
that enable H+/Cl- cotransport also have therapeutic potential. The
immunosuppressive and anticancer activities of the prodigiosin
antibiotics have been attributed to their ability to co-transport H+/
Cl-.3 These promising therapeutic approaches are limited, however,
by the relatively few compounds known to transport Cl- across
membranes.4-6 Herein, we describe the discovery and properties
Figure 1. (A) Schematic representation of pH-stat experiments used to
of a new synthetic Cl- transporter.
monitor H+/Cl- transport. A pH gradient results from NaOH addition to
the extravesicular solution. The charge caused by H+ efflux is compensated
by Cl- efflux, as mediated by the exogenous ligand. The increase in
intravesicular pH, monitored by the entrapped pH-sensitive dye, pyranine,
reflects the electrolyte exchange rate. (B) Initial pseudo-first-order rate
constants in the presence of 5 µM of ionophores 1-6 (1 mol %), obtained
from pH-stat fluorescent assays at room temperature. Suspensions of EYPC
LUVs containing pyranine9 (ex 405 and 460 nm, em 510 nm) in a phosphate
buffer were used (0.5 mM of lipid, 10 mM NanH3-nPO4, n ) 1, 2, pH 6.4,
100 mM NaCl inside and outside). Injection of 20 µL of 0.5 mM DMSO
solution of ionophore 1-6 into 1.9 mL of vesicular suspension was followed
by injection of 21 µL of 0.5 M NaOH.
We recently showed that calix[4]arene tetrabutylamide C1
facilitates H+/Cl- transport in liposomes, forms ion channels in
planar lipid bilayers, and supports electric current in HEK cells.6
A crystal structure of an HCl complex of calix[4]arene tetramethy-
lamide C2 provided a rationale for how Cl- anions are moved
across a membrane by C1. Individual calixarenes were bridged by
amide NH-Cl- and NH-H2O hydrogen bonds to give a lattice
with H2O-filled and Cl--filled pores. The structure revealed that
the calixarene macrocycle was not directly involved in HCl
complexation. We reasoned that ion transport activity might be
maintained, or enhanced, if we dispensed with the calixarene and
produced acyclic analogs that retained the secondary amide groups
needed for Cl- binding and self-association.
had low transport activity at concentrations below 20 µM (4 mol
%). In contrast, application of trimer 3 at concentrations as low as
5 µM (1 mol %) resulted in rapid exchange between extra- and
intravesicular electrolytes (Figure 1B). Trimer 3 was an order of
magnitude more active than C1 at 5 µM (5.8 × 10-2 vs 6.8 ×
10-3 s-1 established previously for C16). Further elongation of the
oligomer backbone resulted in activity decrease. Tetramer 4 was 6
times less active than trimer 3 at 5 µM (1 mol %), and pentamer
5 and hexamer 6 demonstrated further decreases in transport rates.
In an attempt to gain insight into the transport mechanism we
carried out a series of concentration-dependent studies. Whereas
trimer 3 was significantly more active than the other oligomers at
all concentrations, the relative transport abilities of compounds 2-6
varied with concentration, mostly due to the nonlinear increase of
activity observed for dimer 2 (see Figure S2). This nonlinear
increase in transport activity is strong evidence that H+/Cl- transport
is mediated by a self-associated form of 2. However, the apparent
linear concentration-activity relationship observed for trimer 3
suggests that trimer 3 is either pre-assembled before membrane
insertion or that it acts by a carrier mechanism.8
To test our hypothesis, we synthesized the series of phenoxy-
acetamides 1-6,7 ranging from monomer 1 to hexamer 6 and
evaluated their ability to support H+/Cl- transport in large unila-
mellar vesicles (LUVs). We report that trimer 3 is the most efficient
chloride transporter among compounds 1-6 and calix[4]arene C1.
Trimer 3 also has an unprecedented function for a synthetic
compound. Due to its high Cl-/SO42- transport selectivity, 3 induces
a stable potential in liposomes experiencing a transmembrane Cl-/
SO42- gradient. These results represent encouraging developments
in the search for new classes of synthetic Cl- transporters.
The H+/Cl- transport activity of 1-6 was evaluated via the pH-
stat fluorescent assay (Figure 1).6 A significant increase in transport
rates with increasing polymer chain length was observed for
compounds 1-3. Monomer 1 showed essentially no transport
activity, even at 50 µM (10 mol %, ligand-to-lipid ratio). Dimer 2
Importantly, compounds 2-6 mediated electrolyte exchange in
2-
the presence of Cl- but not in the presence of SO4 anion. In
contrast to the results shown in Figure 1B, where NaCl extra- and
intravesicular buffers were used, no transport activity was detected
in LUVs symmetrically loaded with Na2SO4, even at ligand
concentrations of 50 µM (10 mol %). This anion-dependent activity
is strong evidence that butylamides 2-6 mediate Cl- transport
across the bilayer.
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10.1021/ja029372t CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
outside). The lower two traces in Figure 2B verify that trimer 3
and valinomycin are functionally orthogonal. Trimer 3 creates a
transmembrane potential under conditions where valinomycin
cannot create a potential and vice versa.
To our knowledge, trimer 3 is the first compound to induce a
stable potential in LUVs due to a transmembrane anionic gradient.13
These data show that trimer 3 has a significant anion/cation transport
selectivity. Maintenance of this potential and the stable transmem-
brane Cl- equilibrium demonstrated by 35Cl NMR (Figure 2) also
indicate that trimer 3 does not induce membrane defects. The ability
of trimer 3 to transport Cl-, to generate and maintain a transmem-
brane potential, along with its high activity at low µM concentra-
tions, its low molecular weight, and its simple preparation, make
this compound a potentially valuable lead in drug development for
the treatment of cystic fibrosis and cancer.2,3,14 We do not know
yet whether this chloride transporter functions as a channel or as a
carrier. The mechanism by which these oligomers, particularly
trimer 3, transport Cl- across membranes is a major focus of our
ongoing research.
Figure 2. (A) 35Cl NMR spectra of a suspension of giant vesicles (88 mM
EYPC, 9:1 H2O:D2O, 100 mM NaCl, 10 mM CoCl2,10 mM NanH3-nPO4,
n ) 1, 2, pH 5.4) suspended in 75 mM Na2SO4 Co2+-free buffer (9:1 H2O:
D2O, NanH3-nPO4, n ) 1, 2, pH 6.4). Spectra correspond to (a) giant vesicles
in the absence of 3, (b) giant vesicles 1 h after application of 1 mol % 3 in
DMSO, and (c) vesicles after lysis with Triton X-100. (B) Liposome
potential fluorescent assays. Suspension of EYPC LUVs at room temperature
was used (10 mM NanH3-nPO4, n ) 1, 2, pH 6.4, 100 mM KCl or 75 mM
Na2SO4 inside and 100 mM NaCl, 60 nM potential-sensitive dye safranin
O, ex 480 nm, em 520 nm, outside). Color code for traces denotes the
formation of potential in: (blue) KCl vesicles upon application of
valinomycin, (orange) Na2SO4 vesicles upon application of 3, (red) KCl
vesicles upon application of 3, (green) Na2SO4 vesicles upon application
of valinomycin. Potentials were quenched at the end of each experiment
by injecting 20 µL of a 1 mM aqueous solution of the defect-inducing
peptide melittin.
Acknowledgment. We thank the Department of Energy for
support. J.T.D. is a Dreyfus Teacher-Scholar. F.W.K. thanks the
ACS for a Division of Organic Chemistry Graduate Fellowship
sponsored by AstraZeneca. We thank Lyle Isaacs for comments,
Simi Adeyeye for assistance, and Neil Blough for use of his
fluorimeter.
Direct evidence for Cl- transport by trimer 3 was obtained from
35Cl NMR experiments. Giant vesicles containing NaCl and CoCl2
were suspended in Co2+-free Na2SO4 buffer. The membrane-
impermeable Co2+ caused a downfield shift and broadening of the
35Cl NMR signal for intravesicular Cl-.10 A separate, smaller signal
was due to residual extravesicular Cl- (Figure 2A,a). Controls
showed no leakage of Cl- from liposomes even after 3 days.
Addition of trimer 3 resulted in an increased extravesicular Cl-
peak due to outwardly directed Cl- transport (Figure 2A,b). The
new intravesicular/extravesicular Cl- equilibrium in the presence
of 3 was stable for at least 3 h, or until lysis with Triton X-100
released the intravesicular Cl- to give a single 35Cl NMR resonance
(Figure 2A,c).11
Supporting Information Available: Synthetic preparations and
experimental details (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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The combined pH-stat and 35Cl NMR data indicate that the
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Although trimer 3 transports both Cl- and H+ into sulfate-loaded
liposomes suspended in a chloride buffer, Cl- is transported faster
than H+, and therefore the overall process is not electrically silent.
Monitoring of transmembrane potential using the potential-sensitive
dye safranin O12 revealed the formation of stable negative charge
inside liposomes (75 mM Na2SO4 inside, 100 mM NaCl outside,
safranin O outside) within 2 min of applying 1 mol % of trimer 3
(Figure 2B, red trace). The magnitude of the potential induced by
3 under an inwardly directed Cl- gradient is similar to that generated
by 0.12 mol % valinomycin in liposomes with an outward K+
gradient (Figure 2B, blue trace, 100 mM KCl inside, 100 mM NaCl
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