Large and stable transmembrane pores from guanosine-bile acid conjugates

Article abstract of DOI:10.1021/ja7110702

We report that a ditopic guanosine-lithocholic acid conjugate forms discrete channels in planar phospholipid membranes. These pores are large, with nanoSiemens conductance levels, and stable, with "open" times of seconds, distinguishing them from most synthetic channels, which typically conduct in the picosiemens range with millisecond lifetimes. We propose that these large pores are due to the formation of G4-quartet based superstructures within the membrane. Copyright

Full text of DOI:10.1021/ja7110702

Published on Web 02/15/2008
Large and Stable Transmembrane Pores from Guanosine-Bile Acid
Conjugates
Ling Ma,† Monica Melegari,† Marco Colombini,‡ and Jeffery T. Davis*,†
Department of Chemistry and Biochemistry and Department of Biology, UniVersity of Maryland,
College Park, Maryland 20742
Received December 18, 2007; E-mail: jdavis@umd.edu
A variety of natural and synthetic compounds are known to self-
assemble to give transmembrane ion channels.1 Hydrogen-bonded
macrocycles that can π-stack are a new type of channel motif.2
Thus, folate quartets stack to give ion channels in lipid bilayers.3
This folate assembly had a single-channel conductance of 10-20
picosiemens (pS), values consistent with the quartet’s 3 Å diameter.
We found that a noncovalent assembly of 16 guanosine monomers
could be cross-linked to give a “unimolecular” G-quadruplex that
can transport Na+ across lipid membranes.4
We now report that the ditopic guanosine-lithocholate 1 forms
discrete channels in phospholipid membranes (Figure 1). These
pores are large (nS conductance) and stable, with “open” times of
seconds, distinguishing them from most synthetic channels, which
typically conduct in the pS range with millisecond lifetimes.1,5
Lehn and Barboiu have independently shown that ditopic
monomers with guanine end groups form supramolecular polymers
in cation-templated processes.6 Possible supramolecular structures
built from these ion-templated G4-quartets are depicted in Figure
2. In addition to the G4-quartet channel, such structures might well
stack to form pores for transmembrane transport. Of relevance was
Barboiu’s demonstration that Na+ and K+ could be transported
across films made from G4-quartet polymers.6b
Figure 1. Ditopic G-sterol 1 and control bis-lithocholamide 2. Typical
traces of conductance vs time, after addition of 1 or 2, are depicted. The
guanosine end-groups are needed to form a transmembrane channel.
The nucleoside-sterol conjugate 1 has two guanosine groups
connected by a bis-lithocholate linker. This spacer was inspired by
Kobuke’s studies that showed that bis-cholic acid derivatives formed
cation-selective channels with pS conductance.7,8 We envisioned
that membrane insertion of 1, followed by formation of G4-quartets,
might well provide functional pores (Figure 2).
Figure 2. Possible G4-quartet stacks formed by bis-G-lithocholate 1 in a
cation templated process.
Compound 1 was made by coupling 2′,3′-tBDMSi-5′-amino G,9
with a bis-lithocholic acid. Compound 2, with -NMe amide end
groups, was a control. The 1H NMR spectrum of 1 gave sharp peaks
in DMSO-d6, a polar solvent that inhibits self-assembly mediated
by hydrogen bonding. In contrast, the NMR spectrum of 1 in CDCl3
gave much broader signals, consistent with self-association in this
“poor” solvent.
We used CD spectroscopy to gain evidence that 1 forms stacked
G4-quartets in a nonpolar solvent.10 Figure 3 shows CD spectra for
samples of 1 in CHCl3. The CD spectrum of 1 (blue) was taken
after isolation from a silica gel. This sample showed a weak Cotton
band in the 200-280 nm region, suggesting some stacked G4-
quartets.10 We added [2.2.2]-cryptand to ensure that any adventitious
cations bound by 1 were sequestered. Indeed, the resulting spectrum
(green trace) was inactive. We next stirred the mixture of 1 and
[2.2.2]-cryptand in the presence of excess K+ 2,6-dinitrophenolate
(DNP). The CD spectrum of ditopic 1 was much different after
extraction of K+DNP- (red trace). This sample showed a CD
signature diagnostic for stacked G-quartets, with a positive band
at λ ) 266 nm and a negative peak at λ ) 240 nm.10 The complex
Figure 3. CD spectra of guanosine-sterol 1 (0.43 mM) in CHCl3 at room
temperature (blue line), after addition of 1 equiv of [2,2,2]-cryptand (green
line), and after solid-liquid extraction of K+ DNP- (red line). All spectra
were obtained in CHCl3.
formed by 1 and K+ also showed a strong Cotton band at λ ) 295
nm, a signal that has been previously observed for G-quadruplexes
formed from both nucleosides and DNA strands.11,12 The control
compound, NMe amide 2, showed no CD activity under identical
conditions. These data indicate that the K+ ion can template
formation of stacked G4-quartets by 1 in a nonpolar environment.
We next used voltage-clamp experiments to show that 1 forms
single channels in planar membranes; some of these channels had
remarkably long lifetimes and large conductance values for pores
made from a synthetic compound.13 Figure 4 shows representative
records of conductance, as mediated by 1, in a planar bilayer at an
applied voltage of -10 mV in 1 M KCl (trans)/KCl (cis) solution
† Department of Chemistry and Biochemistry.
‡ Department of Biology.
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J. AM. CHEM. SOC. 2008, 130, 2938-2939
10.1021/ja7110702 CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
1 within the phospholipid bilayer. We envision that the bis-
lithocholate linker in 1 provides the walls for the transmembrane
pore and a cation-filled G-quadruplex, formed upon hydrogen-bond
self-assembly, serves as a structural pillar that anchors the assembly
within the membrane.
In summary, ditopic guanosine-sterol 1 forms large and stable
channels. The smaller conductance values near 0.1 pS may arise
as ions are moved through the central channel of a G-quadruplex.3
However, pores that conduct on the 1-20 nS scale must necessarily
have diameters that are significantly larger than that provided by a
G4-quartet. It is tempting to suggest that assemblies like those
depicted in Figure 2, structures previously proposed to explain
formation of G4-quartet polymers,6 are responsible for the function
of 1. Regardless of the actual membrane-active structures, the
demonstration that 1 forms large and stable transmembrane channels
suggests that this nucleoside-sterol may well be able to allow larger
biomolecules to move in and out of liposomes and/or cells. We
are currently pursuing such studies.
Figure 4. Representative traces from voltage-clamp experiments indicating
distinct conductance values recorded in the presence of 1 at -10 mV in 1
M KCl. The number of open events are counted from a total of six
experiments. Three of the experiments were conducted by adding compound
1 (3.1 mM) to the cis side of the chamber after the planar bilayer membrane
was formed (Method 1 in Supporting Information). The other three
experiments were done using membranes that contained compound 1 (0.26
mM) premixed with the phospholipids used to form membrane (Method 2
in Supporting Information).
(pH 7.0). After either application of 1 to the cis side of the planar
bilayer (Method 1) or after premixing compound 1 with the lipid
mixture (Method 2), conductance states of different magnitudes
appeared and disappeared over a 2-3 h period. This pattern of
“open” and “closed” conductance is consistent with dynamic
formation and disintegration of self-assembled channels formed by
1.
Acknowledgment. J.D. thanks the Department of Energy for
support. J.D. and M.C. thank the Maryland Department of Economic
and Business Development for support from the Maryland Nano-
Biotechnology Initiative.
Supporting Information Available: Experimental details and
spectra. This material is available at http://pubs.acs.org
The magnitude and lifetimes of ion conductance supported by 1
varied during a single experiment (Figure 4). Channels with
conductance values of 0.1-1.0 nS typically had the shortest open
lifetimes (10-80 ms). These smaller channels always appeared
during initial events in any experiment. Larger channels with
conductance levels of 1-5 nS had much longer open lifetimes,
typically lasting longer than 10 s. Formation and disassembly of
pores with 1-5 nS conductance were also the most frequent events
observed during any experiment. We occasionally observed long
periods of larger conductance (>20 nS) in the three experiments
that used the Method 1 protocol for introduction of compound 1.
Analysis of data from these six experiments showed similar numbers
of increments and decrements at discrete conductance values,
consistent with the opening and closing of channels of the same
size. The most frequent conductance increments and decrements
were on the order of 1-5 nS. Significantly, addition of control 2
to planar bilayer membranes never resulted in measurable conduc-
tance. Apparently, the guanosine end groups in compound 1 are
essential for pore formation and transmembrane ion transport.
Reversal potentials, recorded in the presence of a 10-fold KCl
gradient, were measured to determine the ion selectivity of the
channels. The reversal potential, Erev, was essentially constant for
the 1-5 nS channels, suggesting no significant enlargement or
contracture of these pores during an experiment. The ion selectivity
of the 1-5 nS channels, calculated from the Goldman-Hodgkin-
Katz equation,14 revealed a cation selective pore (PK+/PCl- ) 6.38
( 0.30). The Hille equation was used to estimate a channel diameter
of ∼2.6-2.7 Å for the smaller channels (0.1 nS at 1 M KCl),5c,13,14
a value that is quite close to the diameter of the G4-quartet’s central
cavity. The pores that conduct in the nS range must, however, be
much larger than a G-quartet. For instance, the Hille diameter for
a single channel of 2.5 nS was estimated to be about 12 Å. Such
a single channel is significantly larger than the diameter of a G4-
quartet, suggesting that ion transport likely proceeds through larger
pore(s) that form upon self-assembly of guanosine-lithocholamide
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