Table 1 Voltage dependence of pore blockage by ADP in sphericala
In summary, the breakthroughs reported in this communication
are (a) experimental evidence for synthetic access to refined pore
architecture with distinct domains, (b) experimental evidence
for voltage-sensitive ‘‘in-depth’’ blockage of synthetic ion channels
and pores1–18 and, from a methodological point of view, (c)
experimental evidence that Woodhull analysis in polarized vesicles
is possible.
and planarb bilayer membranesc
eff c,g
c,h
c,i
zeff
pored zdc,e
zc,f
˚
lW /A
˚
lW/A
1a
2a
3b
a
2
1
1
20.04 ¡ 0.035 21.8
20.22 ¡ 0.008 21.8
20.55 ¡ 0.040 21.8 10.4
0.8
4.2
2.5
12.5
12.5
20.6
20.6
21.5
b
From polarized vesicles (Fig. 2).
From planar bilayer
eff
c
We thank D. Ronan for assistance in synthesis, D. Jeannerat
and A. Pinto for NMR measurements, P. Perrottet and the group
of F. Gu¨lac¸ar for MS measurements, two referees for helpful
comments, and the Swiss NSF for financial support (including the
National Research Program ‘‘Supramolecular Functional
Materials’’ 4047-057496).
conductance (Fig. 3).
experimentally determined Woodhull distance from pore entrance to
From Woodhull eqn (1), lW
5
active site obtained using an assumed blocker charge z, F 5 Faraday
˚
constant, V 5 membrane potential, l 5 pore length (assuming 34 A),
d
temperature. Fig. 1.
e
R
5
gas constant,
T
5
d 5 electric
distance 5 lWeff/l. z 5 assumed charge of ADP at pH 4.5.28
f
g
eff
lW
5 experimentally determined Woodhull distance obtained
h
using z 5 21.8. lW 5 Woodhull distance assumed from molecular
models for detection of rate limiting binding to the first two
i
available RH b-strands.17,18 zeff 5 experimentally determined guest
charge, from eqn (1) using the assumed Woodhull distances lW in the
preceding column.
Notes and references
§ Effective pore concentrations to observe 50% pore activity in the ANTS/
DPX assay under the given conditions (pH 4.5, Fig. 2): EC50 (1) 5 140 nM,
EC50 (2) 5 30 nM.23
were polarized as described previously by connecting a potassium
gradient with the K+-carrier valinomycin.29,22 For inside-negative
polarization, potassium gradients were created by exchanging
external K+ with isoosmolar Na+/K+ mixtures at ratios selected for
the desired Nernst potentials. In double-channel fluorescence
kinetics, changes in the membrane potential were monitored
simultaneously with ANTS/DPX efflux (Fig. 2A, a) following the
emission of the externally added safranin O (Fig. 2A, b). As with
ANTS/DPX efflux, vesicle depolarization by pores 1 and 2 could
be effectively inhibited with ADP (Fig. 2A b, solid vs. dotted),
ATP and IP6. The found increase of the apparent KD for refined
pore 1 but not for classical pore 2 in polarized vesicles was
consistent with blocker repulsion by the inside-negative membrane
potential (Fig. 2C). Interference from stoichiometric binding30 was
likely to account for decreasing voltage sensitivity with excessive
guest charge. Woodhull analysis of the voltage sensitivity of ADP
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eff
˚
revealed an lW 5 4.2 A for the refined pore 1 that was clearly
eff
longer than the nearly negligible lW 5 0.8 A for the classical pore
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˚
2 (Fig. 2D).
eff
˚
The lW 5 4.2 A obtained in polarized vesicles was clearly
eff
shorter than the lW 5 10.4 A obtained in planar bilayer
˚
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conductance experiments (Table 1). The explanation of this
difference with a reduced effective guest charge under these
conditions was supported by the identical zeff 5 20.6 obtained
˚
˚
with the different expected lW 5 2.5 A and lW 5 12.5 A of pores 2
and 1, respectively (Table 1). However, there was no clear-cut
reason for this difference in zeff beyond the general notion that
different effective charges exist under different experimental
conditions (e.g., ionic strength). Partial interference from the
naturally voltage-independent ADP binding in the media was
another meaningful explanation for the apparent underestimation
of Woodhull distances in polarized vesicles. This explanation was
consistent with the intrinsically poor detectability of this possibly
competing process in planar bilayer conductance experiments.
Whatever will turn out to be the reason for the different Woodhull
distances in polarized vesicles and planar bilayer conductance
experiments, we underscore that the essential lesson learned from
the above experiments is that Woodhull analysis of the voltage
dependence of molecular recognition by synthetic multifunctional
pores as such is, at least in a more qualitative sense, possible in
polarized vesicles.
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4800 | Chem. Commun., 2005, 4798–4800
This journal is ß The Royal Society of Chemistry 2005