Chloride Transport Across Vesicle and Cell Membranes by Steroid-Based Receptors

Article abstract of DOI:10.1002/anie.200351957

Crossing the barriers: Membrane transport, a well-established phenomenon for neutral cation receptors such as valinomycin, has been demonstrated for the anion-binding cholapods . The cholapods promote chloride efflux from vesicles by an antiport mechanism and mediate anion flow through live cells grown as epithelia (see schematic representation).

Full text of DOI:10.1002/anie.200351957

Angewandte
Chemie
Membrane Transport
Chloride Transport Across Vesicle and Cell
Membranes by Steroid-Based Receptors**
Atanas V. Koulov, Timothy N. Lambert,
Rameshwer Shukla, Mahim Jain, J. Middleton Boon,
Bradley D. Smith,* Hongyu Li, David N. Sheppard,*
Jean-Baptiste Joos, John P. Clare, and Anthony P. Davis*
It is well-established that molecules which transport cations
across cell membranes (cationophores) can have potent
biological effects.[1] Anion flux is also important to the cell
and, correspondingly, anion carriers may be capable of
biological activity. Indeed, chloride transporters have direct
medical potential as treatments for cystic fibrosis and other
[2]
diseases caused bydefective channel proteins. Despite this
motivation, there have been relativelyfew reports of aniono-
phore natural products[3] or of anion transport bysynthetic
systems.[4] Most, moreover, have involved cationic centers,[5]
which can assist anion passage through the formation of an
ion pair.[4a–g] Anion transport bypurelyelectroneutral systems
is still quite rare.[4j–l] The recentlydescribed “cholapod” anion
Receptors 2 were prepared from cholic acid 3 by
conversion into diamine 4[8] and treatment with appropriate
isocyanates. Initially, their anion-transport properties were
studied byfollowing chloride efflux from unilamellar vesicles
using a chloride-selective electrode. More specifically, 30 mm
receptors 1 are intrinsicallylipophilic, potentiallymembrane-
[6]
soluble, and tuneable to veryhigh affinities.
Certain
cholapods have proved capable of “flippase” activity, that is, unilamellar vesicles (200 nm mean diameter) were prepared
theycan conveypolar head groups across phospholipid
membranes.[7] We therefore supposed that electroneutral
cholapods might transport inorganic anions bya “shuttle”
mechanism, in the manner of cationophores such as valino-
mycin. We now report that cholapods 2 are indeed capable of
transporting chloride ions across liposomal membranes and
also across live cells grown as polarized epithelia.
by extruding a 7:3 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphocholine (POPC) and cholesterol[9] in aqueous NaCl
(500 mm). This stock vesicle dispersion was dialyzed against
aqueous NaNO3 (500 mm) to replace the external chloride
ions with nitrate and then diluted with 500 mm NaNO3 to give
a 1 mm total lipid concentration. As shown in Figure 1,
addition of a solution of 2a–e in THF (4.0 mm final cholapod
concentration) caused chloride efflux from the inner phase of
the vesicles. The initial rates of chloride efflux and the
association constants of 2a–e for Et4N+ClÀ and Et4N+NO3À in
[*] Prof. Dr. B. D. Smith, A. V. Koulov, T. N. Lambert, R. Shukla, M. Jain,
J. M. Boon
Department of Chemistry and Biochemistry and
Walther Cancer Research Center
University of Notre Dame
Notre Dame, IN 46556 (USA)
Fax: (+1)574-631-6652
E-mail: smith.115@nd.edu
Dr. D. N. Sheppard, Dr. H. Li
Department of Physiology
School of Medical Sciences
University of Bristol
University Walk, Bristol BS8 1TD (UK)
Fax: (+44)117-928-8923
E-mail: d.n.sheppard@bristol.ac.uk
Prof. Dr. A. P. Davis, Dr. J.-B. Joos, J. P. Clare
School of Chemistry
University of Bristol
Cantock's Close, Bristol BS8 1TS (UK)
Fax: (+44)117-929-8611
E-mail: anthony.davis@bristol.ac.uk
!
*
Figure 1. Chloride release (r) upon addition of 2e (&), 2d ( ), 2c ( ),
[**] Financial support for this work was provided by the EPSRC (GR/
R04584/01), the University of Bristol, the CF Trust, the NKRF, the
NSF (USA), the Walther Cancer Institute, and the Royal Society.
~
^
2b ( ), and 2a ( ). At time 0 s cholapod (4 mm) was added to vesi-
clesn (1 mm total lipid) containing NaCl (500 mm) dispersed in
NaNO3 (500 mm). The detergent (polyoxythylene(8) lauryl ether) was
added at time 300 s to disrupt the vesicles and release remaining chlo-
ride ions.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2003, 42, 4931 –4933
DOI: 10.1002/anie.200351957
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4931

Communications
Table 1: Initial rates of ClÀ release from vesicles and affinity constants.
efflux altogether. Addition of external NO3À ions to this latter
experiment restored transport (see the Supporting Informa-
tion). These results clearlyindicate an anion antiport
mechanism; substantial chloride efflux can onlyoccur if
there is corresponding influx of an external anion. Cholapod
2d transports both ClÀ and NO3À ions, but is less effective for
Compound Initial rate of
efflux [% sÀ1 [a]
Ka
Ka (Et4N+-
]
(Et4N+ClÀ) [mÀ1 [b]
]
NO3À) [mÀ1 [b]
]
2a
2b
2c
2d
2e
0.031
0.051
0.075
0.085
0.52
3.4”106
1.2”107
6.3”106
1.5”107[c]
5.2”108
2.1”106
8.8”106
5.1”106
1.0”107[c]
1.7”108
À
the more hydrophilic HCO3 and SO42À ions. In the case of
external sulfate, a small amount of chloride transport
(undetectable using the ClÀ electrode) creates a transmem-
brane potential difference, after which further efflux cannot
occur. In support of this mechanism, cholapod 2d induced an
electric potential (inside negative) across vesicle membranes
partitioning Na2SO4 (inside) and NaCl (outside), as judged by
fluorescence experiments using the potential-sensitive dye
safranin O (see the Supporting Information).[4l]
The abilityof 2e to transport chloride ions into vesicles
was detected byusing 35Cl NMR spectroscopy. In this case,
vesicles were formed in 300 mm NaBr solution and then
dialyzed against aqueous NaCl (300 mm) containing CoCl2
(10 mm) as a 35Cl NMR shift reagent.[4a,10] Internalized
chloride ions were detected as an unshifted 35Cl NMR
resonance after addition of 2e (Figure 3). Separate resonance
peaks for the internal and external chloride ions were
observed for at least 24 h, thus showing that the vesicle
membranes remain unperturbed bythe cholapod.
[a] For details see text and legend to Figure 1. [b] Average of two
measurements. [c] Determinations made on the corresponding eicosyl
ester to avoid overlap of NMR signals.
water-saturated CHCl3, as measured byextraction, are listed
in Table 1.[6b] The efflux rates correlate reasonablywell with
the association constants, thus indicating that a hydrogen-
bonding recognition event controls the transport process. As
expected, compounds 5 and 6 (Boc = tert-butoxycarbonyl)
with veryweak chloride affinityproduced no measurable
efflux.
In principle, cholapods 2 might act as mobile carriers or
through formation of multicomponent channels. To date,
there are two pieces of evidence suggesting the former. First,
Further studies were performed on 2d to clarifythe
mechanism of transport. Firstly, chloride efflux was shown to
be unaltered upon changing the entrapped countercation
from sodium to either potassium or caesium (see the
Supporting Information), which indicates that the metal
cation is not involved in the transport process. Secondly, the
external anion was varied. As shown in Figure 2, the effect
was substantial. For example, replacement of the external
À
NO3 with HCO3À ions lowered transport rates byabout
80%, while substitution with SO42À ions prevented chloride
Figure 3. 35Cl NMR spectra of vesicles (30 mm total lipid) containing
300 mm NaBr dispersed in a solution of 300 mm NaCl and 10 mm
CoCl2. Spectra were obtained a) before and b) 24 h after addition of 2e
(4 mm). Signals corresponding to chloride ions outside and inside the
vesicles are labeled accordingly.
Figure 2. Dependence of the chloride release (r) induced by 2d on
external anions. The vesicles containing NaCl (500 mm) were dis-
!
*
persed in isotonic NaNO3 (&), NaHCO3 ( ), or Na2SO4 ( ). See
Figure 1 for other details.
4932
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2003, 42, 4931 –4933

Angewandte
Chemie
Merritt, M. Lanier, G. Deng, S. L. Regen, J. Am. Chem. Soc.
efflux rates with 2d increased just 3.2-fold when the receptor
concentration was increased from 4 to 40 mm (see the
Supporting Information). This less than first-order depen-
dence suggests the formation of inactive, rather than active,
aggregates at the higher concentration.[11] Second, there is no
cholapod-mediated efflux of chloride ions from vesicles
composed of dipalmitoylphosphatidylcholine (DPPC) at
room temperature (gel phase), but facilitated efflux occurs
above the temperature for the gel/liquid crystalline phase
transition (fluid phase). This is signature behavior of a mobile
carrier (for example, monensin) whose activitydepends on
membrane viscosity; in contrast, channel activity should not
show this effect.[12]
1998, 120, 8494; f) N. Sakai, S. Matile, Tetrahedron Lett. 1997, 38,
2613; g) B. Dietrich, T. M. Fyles, M. W. Hosseini, J.-M. Lehn,
K. C. Kaye, J. Chem. Soc. Chem. Commun. 1988, 691; h) D. P.
Wallace, J. M. Tomich, J. W. Eppler, T. Iwamoto, J. J. Grantham,
L. P. Sullivan, Biochim. Biophys. Acta 2000, 1464, 69; i) K. E.
Mitchell, T. Iwamoto, J. Tomich, L. C. Freeman, Biochim.
Biophys. Acta 2000, 1466, 47; j) P. H. Schlesinger, R. Ferdani, J.
Liu, J. Pajewska, R. Pajewski, M. Saito, H. Shabany, G. W.
Gokel, J. Am. Chem. Soc. 2002, 124, 1848; k) V. Sidorov, F. W.
Kotch, G. Abdrakhmanova, R. Mizani, J. C. Fettinger, J. T.
Davis, J. Am. Chem. Soc. 2002, 124, 2267; l) V. Sidorov, F. W.
Kotch, J. L. Kuebler, Y. F. Lam, J. T. Davis, J. Am. Chem. Soc.
2003, 125, 2840.
[5] Present in the transporter itself or in cotransported counter-
cations.
Finally, cholapod-mediated ClÀ transport was demon-
strated in live cells byusing Madin Darbycanine kidney
(MDCK) epithelia and the Ussing chamber technique.[13] The
method involves growing an oriented layer of cells on a filter
support and placing it between current- and voltage-measur-
ing electrodes. Endogenous active transport systems generate
a potential difference across the cell membranes. Addition of
a passive chloride transporter causes discharge of the electro-
chemical gradient, which is detected as a flow of current.
Receptor 2e (80 mm) did indeed produce this effect (see the
Supporting Information). As expected, the response was
anion-dependent; replacement of chloride bygluconate
anions on either side of the membrane reduced the signal
byabout 80%. Moreover, the response was different from
that induced byactivators of native chloride channels found in
MDCK epithelia (see the Supporting Information).
In conclusion, cholapods 2 have been shown bythree
techniques (electrochemical, NMR, and fluorescence) to
transport chloride ions across vesicle membranes. The data
favors an antiport mechanism with the cholapod more likely
acting as a mobile carrier than a channel. The cholapod does
not disrupt the bilayer membrane and is able to convert a
chloride concentration gradient into a transmembrane elec-
trical potential. Facilitated chloride transport has also been
demonstrated in polarized epithelial cells, which augurs well
for future applications in biochemistryand medicine.
[6] a) A. P. Davis, J. J. Perry, R. P. Williams, J. Am. Chem. Soc. 1997,
119, 1793; A. J. Ayling, M. N. Pꢀrez-Payꢁn, A. P. Davis, J. Am.
Chem. Soc. 2001, 123, 12716; b) A. J. Ayling, S. Broderick, J. P.
Clare, A. P. Davis, M. N. Pꢀrez-Payꢁn, M. Lahtinen, N. J.
Nissinen, K. Rissanen, Chem. Eur. J. 2002, 8, 2197.
[7] T. N. Lambert, J. M. Boon, B. D. Smith, M. N. Pꢀrez-Payꢁn, A. P.
Davis, J. Am. Chem. Soc. 2002, 124, 5276; J. M. Boon, T. N.
Lambert, A. L. Sisson, A. P. Davis, B. D. Smith, J. Am. Chem.
Soc. 2003, 125, 8195.
[8] A. P. Davis, M. N. Pꢀrez-Payꢁn, Synlett 1999, 991.
[9] The 7:3 ratio of POPC to cholesterol mimics that present in
animal cell membranes. Vesicles composed onlyof POPC were
disrupted bythe receptors, as determined bycarboxyfluorescein
leakage assay.
[10] F. G. Riddell in Encyclopedia of Spectroscopy and Spectrometry
(Eds.: J. C. Lindon, G. E. Tranter, J. L. Holmes), Academic
Press, London, 1999, p. 1584.
[11] NMR spectra of 2d in CDCl3 showed concentration dependence,
which confirmed the tendencyfor aggregation in nonpolar
media.
[12] G. Deng, T. Dewa, S. L. Regen, J. Am. Chem. Soc. 1996, 118,
8975.
[13] D. N. Sheppard, M. R. Carson, L. S. Ostedgaard, G. M. Denning,
M. J. Welsh, Am. J. Physiol. 1994, 266, L405.
Received: May22, 2003 [Z51957]
Keywords: anions · membranes · receptors · steroids ·
.
supramolecular chemistry
[1] M. Dobler in Comprehensive Supramolecular Chemistry, Vol. 1
(Ed.: G. W. Gokel), Pergamon, Oxford, 1996, p. 267.
[2] J. R. Broughman, L. P. Shank, W. Takeguchi, B. D. Schultz, T.
Iwamoto, K. E. Mitchell, J. M. Tomich, Biochemistry 2002, 41,
7350, and references therein.
[3] The prodigiosin familyof antibiotics have been shown to be
H+ClÀ cotransporters; for a leading reference, see K. Tanigaki, T.
Sato, Y. Tanaka, T. Ochi, A. Nishikawa, K. Nagai, H. Kawa-
shima, S. Ohkuma, FEBS Lett. 2002, 524, 37.
[4] Leading references: a) A. V. Koulov, J. M. Mahoney, B. D.
Smith, Org. Biomol. Chem. 2003, 1, 27; b) L. A. J. Chrisstoffels,
F. de Jong, D. N. Reinhoudt, Chem. Eur. J. 2000, 6, 1376;
c) L. A. J. Chrisstoffels, F. de Jong, D. N. Reinhoudt, S. Sivelli, L.
Gazzola, A. Casnati, R. Ungaro, J. Am. Chem. Soc. 1999, 121,
10142; d) J. L. Sessler, W. E. Allen, Chemtech 1999, 29, 16; e) M.
Angew. Chem. Int. Ed. 2003, 42, 4931 –4933
www.angewandte.org
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4933