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
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liposomes suspended in a chloride buffer, Cl- is transported faster
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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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(14) Torigoe, T.; Izumi, H.; Ise, T.; Murakami, T.; Uramoto, H.; Ishiguchi,
H.; Yoshida, Y.; Tanabe, M.; Nomoto, M.; Kohno, K. Anticancer Drugs
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