Membrane transporters for anions that use a relay mechanism

Full text of DOI:10.1021/ja8082363

Published on Web 11/26/2008
Membrane Transporters for Anions That Use a Relay Mechanism
Beth A. McNally, Edward J. O’Neil, Anh Nguyen, and Bradley D. Smith*
Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame, Indiana 46556
Received October 20, 2008; E-mail: smith.115@nd.edu
Synthetic bilayer membrane transporters are usually classified
mechanistically as mobile carriers or as ion channels.1 A mobile
carrier associates with a target ion to form a discrete supramolecular
complex that diffuses across the membrane; whereas, an ion channel
is a relatively immobile structure that spans the bilayer and allows
a continuous flow of ions.2 In recent years there has been increased
effort to design synthetic membrane transport systems for anions,
especially Cl-.3 One of the long-term goals of this work is to create
transporter replacement therapies that can alleviate the symptoms
of diseases caused by diminished levels of endogenous Cl- transport
(e.g., cystic fibrosis).4 The field of anion transport is still in its
early stages with most published studies focusing on fundamental
transport studies using model bilayer membranes. In terms of
transporter designs, nearly all have been highly lipophilic com-
pounds that partition strongly and nonselectively into any mem-
brane.5 However, next-generation designs must begin to address
the requirements for pharmaceutical success, including the following
formulation features: (a) acceptable solubility in physiological
solution, (b) appropriate cell targeting and subsequent membrane
partitioning, (c) lengthy residence time in the apical plasma
membrane of target cells. Suitably designed amphiphilic transporters
are likely to exhibit these desirable properties; however, it is quite
challenging to design amphiphilic transporters that operate by carrier
or ion channel mechanisms. This quandary has prompted us to
design a new type of membrane transporter that operates by a relay
mechanism.6
compounds 1-3 were preincorporated into separate samples of
unilamellar vesicles composed of 1-palmitoyl-2-oleoylphosphati-
dylcholine (POPC)/cholesterol (7:3 molar ratio, diameter 200 nm)
and encapsulating the chloride sensitive fluorescent probe lucigenin.
Addition of NaCl to the vesicle dispersions induces Cl- influx and
quenching of the lucigenin fluorescence. The traces in Figure 1
clearly show that transporter 1 is superior to 2, whereas control 3
is essentially inactive. This trend of stronger anionophore producing
enhanced transport has been reported before with a separate class
of mobile carriers for Cl- that also utilize urea groups.13
A generalized picture of the relay transport process is shown in
Scheme 1. The transporter structure is a phospholipid derivative
with an ionophore appended to the end of its sn-2 acyl chain.7 The
ionophore can bind an ion at the membrane surface and then relay
it through the bilayer interior to an acceptor molecule located in
the opposite leaflet.8 In this initial study, the ionophore is a simple
urea group that can associate with Cl- via hydrogen bonds.9 The
phosphatidylcholine derivatives 1-3 were synthesized in a few steps
and high yield using established procedures.10 Transporter 1
contains a 4-nitrophenylurea group with a relatively high affinity
for Cl-, transporter 2 contains a weaker binding 4-tert-butylphe-
nylurea group, and carbamate derivative 3 is a control structure
with very weak Cl- affinity.11
Figure 1. Fluorescence quenching due to Cl- influx. At 100 s, an aliquot
of NaCl (25 mM final concn) was added to vesicles encapsulating the
chloride sensitive probe, lucigenin (1 mM) and NaNO3 (225 mM), T ) 25
°C. The vesicle membranes were composed of POPC/cholesterol (7:3) and
either 1, 2, or 3 (5 mol %).
To elucidate the transport mechanism, a series of additional
experiments were conducted with the most effective transporter,
1. The first experiment demonstrated that replacing the intravesicle
NaNO3 with an isomolar concentration of Na2SO4 produced a
greatly diminished rate of Cl- influx (see Supporting Information,
Figure S1). This is consistent with an anion exchange process; that
is, significant Cl- influx can only occur if there is a corresponding
counteranion efflux, which is greatly diminished with the heavily
The ability of compounds 1-3 to transport Cl- into vesicles was
measured using a standard fluorescence quenching assay.12 Briefly,
Scheme 1. Relay Mechanism for Dimeric Transporter Aggregate
3
2-
solvated SO4
.
The relay mechanism in Scheme 1 implies that the transporter
must reside in both leaflets of the bilayer. This condition was met
in the initial experiment which employed vesicles with 1 prein-
corporated in the membrane. However, no Cl- transport was
observed when the experiment was repeated with one variation,
external addition of 1 to preformed vesicles (see Figure S2 for
details). In this case, the polar lipid 1 inserts into the outer leaflet
of the vesicle membrane (confirmed by UV absorption) and does
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17274 J. AM. CHEM. SOC. 2008, 130, 17274–17275
10.1021/ja8082363 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
not migrate to the inner leaflet. To be effective, transporter 1 needs
to populate both sides of the bilayer membrane.
anionophores appended to the end of the sn-2 acyl chain. The
current design uses urea groups to bind and transport Cl-; however,
it should be possible to employ other molecular recognition units
to produce transporters that are selective for other anions, as well
as cations and neutral polar molecules. Mechanistic studies indicate
that the transporters operate by a new and distinct membrane relay
process. The expected favorable formulation properties of these
amphiphilic compounds (e.g., as liposomes, micelles, etc) should
facilitate efforts to transform them into tools for biomedical research
and perhaps as therapeutic agents.
The dependence of observed Cl- influx rate constants (kobs) on
transporter concentration was determined in two vesicle systems
with membranes of different compositions and thickness (i.e., 1,2-
dimyristoylphosphatidylcholine (DMPC)/cholesterol (7:3) and the
thicker POPC/cholesterol (7:3)). In both cases, the curves were
nonlinear (Figure 2) indicating that transport is mediated by
kinetically active aggregates of 1. Furthermore, linear relationships
are obtained for the two membrane compositions when kobs is plotted
against [1]n, where n ) 2 and 4, respectively.14 Thus, the transporter
aggregate number is two for the DMPC/cholesterol membrane
which is consistent with the slightly overlapped tail-to-tail dimer
shown in Scheme 1. An aggregation of four in the thicker POPC/
cholesterol membrane suggests that a pair of transporters are in
each leaflet as shown in Scheme 2. An increased transporter
aggregation number in thicker membranes has been seen before
with self-assembled pore systems.15
Acknowledgment. This work was supported by the NIH and
the University of Notre Dame.
Supporting Information Available: Synthetic procedures, transport
experiments, and data. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) (a) Stein, W. D. Channels, Carriers and Pumps, An Introduction to
Membrane Transport; Academic: San Diego, CA, 1990. (b) Smith, B. D.;
Lambert, T. N. Chem. Commun. 2003, 2261–2268.
(2) Recent reviews of synthetic transporters: (a) Gokel, G. W.; Carasel, I. A.
Chem. Soc. ReV. 2007, 36, 378–389. (b) Sakai, N.; Mareda, J.; Matile, S.
Mol. Biosyst. 2007, 10, 658–666. (c) Fyles, T. M. Chem. Soc. ReV. 2007,
36, 335–347. (d) McNally, B. A.; Leevy, W. M.; Smith, B. D. Supramol.
Chem. 2007, 19, 29–37. (e) Koert, U.; Al-Momani, L.; Pfeifer, J. R.
Synthesis 2004, 8, 112911–112946.
(3) Davis, A. P.; Sheppard, D. N.; Smith, B. D. Chem. Soc. ReV. 2007, 34,
8–357, and references therein.
(4) Ashcroft, F. M. Ion Channels and Disease; Academic Press: London, 2000.
(5) Notable exceptions are (a) the water soluble peptide transporters designed
by Tomich and coworkers. For a leading reference, see: Shank, L. P.;
Broughman, J. R.; Takeguchi, W.; Cook, G.; Robbins, A. S.; Hahn, L.;
Radke, G.; Iwamoto, T.; Schultz, B. D.; Tomich, J. M. Biophys. J. 2006,
90, 2138–2150. (b) The amphiphilic peptide transporters designed by Gokel
and coworkers. For a leading reference, see: Elliott, E. K.; Stine, K. J.;
Gokel, G. W. J. Membr. Sci. 2008, 321, 43–50.
(6) For studies of relay transport processes in much thicker synthetic plasticized
membranes, see: (a) Riggs, J. A.; Smith, B. D. J. Am. Chem. Soc. 1997,
119, 2765–2766. (b) White, K. M.; Duggan, P. J.; Sheahan, S. L.; Tyndall,
E. M.; Smith, B. D. J. Membr. Sci. 2001, 194, 165–175.
Figure 2. Rate constants (kobs) for Cl- influx at different concentrations
of 1 in vesicles composed of DMPC/cholesterol (7:3) (9) and POPC/
cholesterol (7:3) (b), T ) 25 °C. Inset: linear relationships with [1]n, where
n ) 2 and 4, respectively.
(7) For membrane active phospholipids with simple acyl chain modifications,
see:(a) Menger, F. M.; Aikens, P. Angew. Chem., Int. Ed. 1992, 31, 898–
900. (b) Menger, F. M.; Galloway, A. L.; Chlebowski, M. E.; Wu, S. J. Am.
Chem. Soc. 2006, 128, 14034–14035.
Scheme 2. Relay Mechanism for Transporter Aggregate of Four
(8) It is well established that a modestly polar group appended to the end of
a phospholipid’s acyl chain undergoes substantially dynamic movement
between the lipophilic membrane interior and the polar membrane
surface. (a) Huster, D.; Mu¨ller, P.; Arnold, K.; Herrmann, A. Biophys. J.
2001, 80, 822–831. (b) Loura, L. M. S.; Ramalho, J. P. P. Biochim. Biophys.
Acta 2007, 1768, 467–478. (c) Menger, F. M.; Keiper, J. S.; Caran, K. L.
J. Am. Chem. Soc. 2002, 124, 11842–11843.
(9) 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. Chem.sEur. J. 2008, 14, 9599–9606.
The final mechanistic study with 1 measured Cl- influx rates as
a function of vesicle membrane thickness. Transport was monitored
in vesicles composed of phospholipids with increasing acyl chain
length, and thus increased membrane thickness.15,16 Figure S3
shows that increasing the acyl chain carbon number from 14 to 18
produced an incremental decrease in transport rate. Significantly,
when the acyl carbon number was increased to 20 and above there
was a dramatic drop to essentially zero transport. This membrane
thickness threshold effect is consistent with the relay mechanism
and not with the two alternatives.17 When the membrane is
relatively thin, the transporters can effectively relay Cl- across the
lipophilic core of the membrane as shown in Schemes 1 and 2.
Once the membrane is thicker than the tail-to-tail aggregate in
Scheme 2 (whose polar head groups are anchored to their respective
membrane interfaces), there is a gap between the urea groups in
each leaflet and the energetic barrier for Cl- relay becomes
prohibitively high.
(10) (a) Roseto, R.; Hajdu, J. Tetrahedron Lett. 2005, 46, 2941–2944. (b)
Lampkins, A. J.; O’Neil, E. J.; Smith, B. D. J. Org. Chem. 2008, 73, 6053–
6058.
(11) The Cl- association constant for N-(4-nitrophenyl)-N′-octyl urea in water
saturated chloroform is ∼8000 M-1 and ∼5000 M-1 for N-(4-tert-
butylphenyl)-N′-octyl urea as determined by the method in ref 9.
(12) McNally, B. A.; Koulov, A. V.; Smith, B. D.; Joos, J. B.; Davis, A. P.
Chem. Commun. 2005, 1087–1089.
(13) Koulov, A. V.; Lambert, T. N.; Shukla, R.; Jain, M.; Boon, J. M.; Smith,
B. D.; Li, H. Y.; Sheppard, D. N.; Joos, J. B.; Clare, J. P.; Davis, A. P.
Angew, Chem., Int. Ed. 2003, 42, 4931–4933.
(14) Otto, S.; Osifchin, M.; Regen, S. L. J. Am. Chem. Soc. 1999, 121, 7276–
7277.
(15) DiGiogio, A. F.; Otto, S.; Bandyopadhyaya, P.; Regen, S. L. J. Am. Chem.
Soc. 2000, 122, 11029–11030.
(16) Lewis, B. A.; Engelman, D. M. J. Mol. Biol. 1983, 166, 211–217.
(17) Membrane thickness studies with a classic mobile carrier like valinomycin
report a moderate ten-fold decrease in transport rate as the acyl chain carbon
number is increased from 16 to 22 (Benz, R.; Frohlich, O.; La¨uger, P.
Biochim. Biophys. Acta 1977, 464, 465–481. With channel transport
processes there is a bell-shaped relationship with membrane thickness;
optimal transport is observed when the membrane thickness matches the
length of the channel structure: Weber, M. E.; Schlesinger, P. H.; Gokel,
G. W. J. Am. Chem. Soc. 2005, 127, 636–642.
In summary, we report a new class of synthetic membrane
transporters whose molecular structures are phospholipids with
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