Optical switching of ion-dipole interactions in a gramicidin channel analogue

Article abstract of DOI:10.1021/ja000736w

Optical control of ion channel gating could permit the functional manipulation of excitable cells. We wished to examine the feasibility of using optical switching of ion-dipole interactions as a means of switching ion flux in channels. We prepared an anal

Full text of DOI:10.1021/ja000736w

6364
J. Am. Chem. Soc. 2000, 122, 6364-6370
Optical Switching of Ion-Dipole Interactions in a Gramicidin
Channel Analogue
Vitali Borisenko, Darcy C. Burns, Zhihua Zhang, and G. Andrew Woolley*
Contribution from the Department of Chemistry, UniVersity of Toronto, 80 St. George Street,
Toronto, Ontario M5S 3H6, Canada
ReceiVed February 29, 2000
Abstract: Optical control of ion channel gating could permit the functional manipulation of excitable cells.
We wished to examine the feasibility of using optical switching of ion-dipole interactions as a means of
switching ion flux in channels. We prepared an analogue of the ion channel gramicidin A in which an azobenzene
side chain was substituted for a valine side chain at position 1. The dipole moment of the azobenzene group
can be reversibly switched between approximately 3 and 0 D by cis-trans photoisomerization. The observed
conductance properties of the modified channels can be understood in terms of (switchable) ion-dipole
interactions that control the height of the central barrier for Cs+ and Na+ movement through the pore. The
predictable behavior of the system implies that larger dipole changes or changes closer to the central axis of
the pore might effect complete gating.
Introduction
relative simplicity makes chemical modification straightforward.
Gramicidin dimers are formed by the association of two single-
stranded â-helices, joined by intermolecular hydrogen bonds
at their amino termini.19,20 Several photosensitive gramicidin
analogues have been synthesized.13,16,17,21 Each of these em-
ployed an azobenzene derivative as the photochromic unit. This
group is robust and undergoes reversible cis-trans photo-
isomerization without significant photobleaching.22 The wave-
lengths required (>330 nm) do not cause photodegradation of
gramicidin Trp residues.23
Optical methods have demonstrated utility for the investiga-
tion of physicochemical properties of biochemical systems.1-7
A photoregulated ion channel would extend the range of these
investigations by enabling the direct manipulation of cellular
excitability using light. A photoregulated ion transport system
might, in principle,8-10 involve photoregulation of ion-binding
sites,11 positioning of channel-forming molecules in the mem-
brane,12,13 creation of polar sites within the membrane,14 or direct
alteration of the properties of ion channels.10,15-17
Optical modulation of the insertion of a gramicidin into
bilayers has been reported with a C-terminal azobenzene-based
modification.13 Covalently linked dimers of gramicidin have also
been reported, in which an azobenzene group forms part of the
linker.16,17 Although effects of photoisomerization on channel
activity were observed in these cases, a straightforward inter-
pretation of activity in structural terms was not possible.
Previously, we reported the modification of the C-terminal end
of gramicidin with an alkylamino-azobenzene moiety. This
derivative showed photosensitive blocking of the entrance and
exit of the gramicidin channel.21,24 Although the photomodu-
lation could be understood in structural terms, significant
redesign of the C-terminus of the peptide would be required
for very large changes in conductance to be obtained.
The gramicidin channel is an attractive target for rational
design because of its well-defined structural and functional
properties under a wide range of conditions.18 In addition, its
* Corresponding author. Tel./fax: 416-978-0675. E-mail: awoolley@
chem.utoronto.ca.
(1) Willner, I.; Rubin, I. Angew. Chem., Int. Ed. Engl. 1996, 35, 367-
385.
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117, 8466-8467.
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U.S.A. 2000, 97, 3803-3808.
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(5) Nerbonne, J. M. Curr. Opin. Neurobiol. 1996, 6, 379-386.
(6) Politz, J. C. Trends Cell. Biol. 1999, 9, 284-287.
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88.
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Springer-Verlag: Berlin, 1990; Vol. 1, pp 161-195.
(12) Ueda, T.; Nagamine, K.; Kimura, S.; Imanishi, Y. J. Chem. Soc.,
Perkin Trans. 2 1995, 365-368.
In view of recent results supporting an important role for ion-
dipole interactions in controlling conductance in gramicidin
channels25-29 and in cation channels generally,30,31 we wished
(19) Andersen, O. S. Annu. ReV. Physiol. 1984, 46, 531-548.
(20) Woolley, G. A.; Wallace, B. A. J. Membr. Biol. 1992, 129, 109-
136.
(21) Lien, L.; Jaikaran, D. C. J.; Zhang, Z.; Woolley, G. A. J. Am. Chem.
Soc. 1996, 118, 12222-12223.
(22) Rau, H. In Photochromism. Molecules and Systems; Durr, H., Bouas-
Laurent, H., Eds.; Elsevier: Amsterdam, 1990; pp 165-192.
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78.
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A. V. Biophys. J. 1997, 73, 2465-2475.
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D.; Koeppe, R. E., II. J. Am. Chem. Soc. 1998, 120, 5142-5146.
(13) Osman, P.; Martin, S.; Milojevic, D.; Tansey, C.; Separovic, F.
Langmuir 1998, 14, 4238-4242.
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(15) Chang, C. Y.; Niblack, B.; Walker, B.; Bayley, H. Chem. Biol. 1995,
2, 391-400.
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Biophys. Acta 1991, 1061, 163-170.
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Arseniev, A. S. Biol. Membr. 1993, 10, 535-542 (in Russian).
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10.1021/ja000736w CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/22/2000

Optical Switching of Ion-Dipole Interactions
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6365
Figure 1. Chemical structure of phenylazo-phenylalanine (Pap1)-gramicidin A. (Inset) Pap side-chain torsion angle designations.
to test the possibility of channel photoregulation via dipole
switching. Detailed studies of the effects of side-chain dipoles
on the function of gramicidin channels have been performed
by several groups.25,29,32,33 Even though gramicidin side chains
are not in direct contact with the permeating ion, channel
properties can be strongly affected by the position and magnitude
of side-chain dipole moments. Efforts have been focused on
understanding the effects of Trp side-chain dipole moments on
conductance (at positions Trp9, Trp11, Trp13, Trp15)26-29,34,35 and
the effects of side-chain dipole moment changes at position 1
of the channel (in the center of the membrane).36-39 Given the
well-defined structure of the gramicidin channel, the effects of
these dipoles can be discussed with reference to specific
orientations of side-chain dipoles relative to the ion permeation
pathway. The results of all these studies have been rationalized
in terms of through-space ion-dipole interactions.25,29,32,33,36
We decided to focus on dipole switching at position 1, since,
under many conditions, ion translocation is rate limiting in the
gramicidin channel so that electrostatic changes near the center
of the membrane might be expected to have the largest effects
on conductance.19,36 For instance, the single-channel sodium
conductance decreased 10-fold when hexafluorovaline (side-
chain dipole, +1.6 D) was substituted for valine (side-chain
dipole, -0.4 D) at position 1.37
There is a significant dipole moment change associated with
trans-to-cis isomerization of azobenzene (0-3 D).22 We there-
fore prepared a gramicidin derivative in which valine at position
1 was replaced by phenylazo-phenylalanine (Pap, Figure 1). We
find that photoisomerization of Pap1-gA(trans) to Pap1-gA(cis)
causes conductance changes that can be understood in terms of
an increase in the central barrier for Cs+ and Na+ translocation
via an ion-dipole interaction.
Materials and Methods
N-Fmoc-p-amino-phenylalanine (Fmoc ) 9-fluorenylmethyl-oxy-
carbonyl) was obtained from Bachem (King of Prussia, PA). Di-
phytanoyl-phosphatidylcholine was purchased from Avanti Polar Lipids
Inc. (Alabaster, AL). Gramicidin, decane, CsCl, NaCl, HCl, TEA, and
the buffer N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)
were obtained from Sigma Aldrich Canada (Oakville, ON).
Synthesis. The preparation of gramicidin in which the valine at
position 1 is replaced by phenylazo-phenylalanine (Pap1-gA) followed
the semisynthetic procedure described by Weiss and Koeppe40 and
Morrow et al.38,41 for the preparation of other position 1 mutants of
gramicidin.
(26) Busath, D. D.; Thulin, C. D.; Hendershot, R. W.; Phillips, L. R.;
Maughan, P.; Cole, C. D.; Bingham, N. C.; Morrison, S.; Baird, L. C.;
Hendershot, R. J.; Cotten, M.; Cross, T. A. Biophys. J. 1998, 75, 2830-
2844.
(27) Cotten, M.; Tian, C.; Busath, D. D.; Shirts, R. B.; Cross, T. A.
Biochemistry 1999, 38, 9185-9197.
(28) Dorigo, A. E.; Anderson, D. G.; Busath, D. D. Biophys. J. 1999,
76, 1897-1908.
(i) Preparation of Fmoc-Pap. Fmoc-protected Pap was prepared
via a modification of the procedure we described previously.42 N-Fmoc-
p-amino-phenylalanine (1.9 g, Bachem) was combined with 1.5 equiv
of nitrosobenzene (Aldrich) in 150 mL of glacial acetic acid and stirred
at room temperature overnight. The reaction mixture was poured into
900 mL of cold water, and the pH was adjusted to 4 with sodium
bicarbonate. The brown precipitate was washed with water and then
redissolved in 90 mL of THF. The solution was cooled on ice, and
cold water was added slowly (approximately 60 mL) until a precipitate
formed. This procedure was repeated two more times to yield 1.1 g of
Fmoc-Pap as a yellow-orange solid (yield 47%). TLC (EtOAc/PE/
MeOH 3:2:2.8): Rf ) 0.7. High-resolution FABMS: MH+ C30H26N3O4,
calcd 492.1923, found 492.1928.
(29) Phillips, L. R.; Cole, C. D.; Hendershot, R. J.; Cotten, M.; Cross,
T. A.; Busath, D. D. Biophys. J. 1999, 77, 2492-2501.
(30) Chung, S. H.; Allen, T. W.; Hoyles, M.; Kuyucak, S. Biophys. J.
1999, 77, 2517-2533.
(31) Roux, B.; MacKinnon, R. Science 1999, 285, 100-102.
(32) Benamar, D.; Daumas, P.; Trudelle, Y.; Calas, B.; Bennes, R.; Heitz,
F. Eur. Biophys. J. 1993, 22, 145-150.
(33) Hu, W.; Cross, T. A. Biochemistry 1995, 34, 14147-14155.
(34) Becker, M. D.; Greathouse, D. V.; Koeppe, R. E., II; Andersen, O.
S. Biochemistry 1991, 30, 8830-8839.
(ii) Preparation of Fmoc-Pap1-Gramicidin. Fmoc-Pap (15.2 mg)
was mixed with 1 equiv of O-(7-azabenzotriaol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HATU) and 2 equiv of
(35) Daumas, P.; Benamar, D.; Heitz, F.; Ranjalahy-Rasoloarijao, L.;
Mouden, R.; Lazaro, R.; Pullman, A. Int. J. Pept. Protein Res. 1991, 38,
218-228.
(36) Koeppe, R. E., II; Mazet, J. L.; Andersen, O. S. Biochemistry 1990,
29, 512-520.
(40) Weiss, L. B.; Koeppe, R. E., II. Int. J. Pept. Protein Res. 1985, 26,
305-310.
(37) Barrett Russell, E. W.; Weiss, L. B.; Navetta, F. I.; Koeppe, R. E.,
II; Andersen, O. S. Biophys. J. 1986, 49, 673-686.
(38) Morrow, J. S.; Veatch, W. R.; Stryer, L. Biophys. J. 1978, 17, 26a.
(39) Russell, E. W.; Weiss, L. B.; Navetta, F. I.; Koeppe, R. E., II;
Andersen, O. S. Biophys. J. 1986, 49, 673-686.
(41) Morrow, J. S.; Veatch, W. R.; Stryer, L. J. Mol. Biol. 1979, 132,
733-738.
(42) Liu, D.; Karanicolas, J.; Yu, C.; Zhang, Z.; Woolley, G. A. Bioorg.
Med. Chem. Lett. 1997, 7, 2677-2680.

6366 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Borisenko et al.
diisopropylethylamine (DIPEA) in 1.2 mL of dry DMF, and then the
mixed solution was added dropwise to 49 mg (0.9 equiv) of des-
(formylvalyl)-gramicidin (prepared exactly as described in ref 40) over
5 min. The reaction mixture was stirred at room temperature for 3 h,
and then the solvent was removed under high vacuum. The product
was dissolved in 1 mL of methanol and loaded onto an LH-20 gel
filtration column running in methanol. The fractions containing product,
as identified by their UV spectra, were combined, and the methanol
was removed to give Fmoc-Pap1-gramicidin as an orange solid. Rf )
0.8 (chloroform/methanol/water (CMW) 65:25:4). MS(ES): [MH+]
C
123H155N22O18, calcd 2229.7, obsd 2229.
(iii) Preparation of Pap1-Gramicidin. Fmoc-Pap1-gramicidin (65.9
mg) was dissolved in 0.8 mL of dry DMF, and then 1.5 mL of 20%
piperidine in DMF was added dropwise. The reaction mixture was
stirred at room temperature overnight, and then DMF and piperidine
were removed under high vacuum. The solid was dissolved in 1 mL
of MeOH and again loaded onto an LH-20 gel filtration column. The
fractions containing product, as identified by their UV spectra, were
pooled and concentrated to give 30 mg of a yellow solid. Rf ) 0.78
(CMW 65:25:4). MS(ES): [MH+] C108H145N22O16, calcd 2007.5, obsd
2007.
Figure 2. UV spectra of dark-adapted (trans) Pap1-gA (- - -) and
photoirradiated Pap1-gA (s) in methanol.
(iv) Preparation of Formic-Acetic Anhydride. Formic acid (2.5
mL) was added dropwise to 5 mL of cooled acetic anhydride with
stirring. The reaction mixture was heated to 50 °C for 15 min with
stirring and then allowed to cool and stored in a refrigerator.
(v) Preparation of N-Formyl-Pap1-Gramicidin. Pap1-gramicidin
(30 mg) was dissolved in 2 mL of THF and cooled in ice water. The
pH of the reaction mixture was adjusted from 5 to 7 by adding 1 drop
of TEA, and then 25 µL of formic-acetic anhydride was added. The
reaction mixture was allowed to warm to room temperature and then
stirred at room temperature for 2 h. The solvent was removed under
high vacuum to give an orange product that was redissolved in 1 mL
of methanol and then purified twice by HPLC. The HPLC conditions
were as follows: Zorbax C-8 column, isocratic conditions, 88%
methanol/12% water, flow rate 1.2 mL/min (retention time 6 min).
Rf ) 0.76 (CMW 65:25:4). MS(ES): [MH+] C109H145N22O17, calcd
2035.5, obsd 2034.9; MNa+ 2056.8. Purity as estimated by HPLC was
greater than 98%.
Single-Channel Measurements. The general techniques for single-
channel recording of gramicidin channels have been described previ-
ously.43 Peptides (∼10 nM in MeOH) were added to membranes formed
from diphytanoyl-phosphatidylcholine/decane (50 mg/mL). The trans
state of Pap was obtained by dark adaptation of a stock solution of the
peptide in MeOH. Photoisomerization to the cis state was achieved by
irradiation of the stock solution with a nitrogen laser (λ ) 337 nm;
150 µJ/pulse; 1.5 ns/pulse/15 Hz) for ∼1 h. Although isomerization in
situ is also possible with our experimental arrangement,21 isomerization
of stock solutions was performed to permit monitoring of the cis-
azobenzene content by UV absorbance spectroscopy. Lipid bilayers
were formed across a ∼100-µm hole in a polypropylene pipet tip by
painting a solution of lipid in decane. The pipet tip was mounted in a
Teflon cell through a small hole in the back face. The front face of
this cell had a removable circular glass window. Silver/silver chloride
electrodes were placed in the pipet tip and in a cylindrical well drilled
from the top of cell. Symmetrical, buffered (5 mM BES, pH 7) CsCl
(1 or 0.1 M), NaCl (1 M), or HCl (0.1 M, unbuffered) solutions were
used. All measurements were made at room temperature.
The gramicidin channel structure, taken as a starting point for
conformational energy and electrostatic calculations, was obtained from
the Protein Data Bank (PDB code 1MAG). A single Na+ ion was
positioned in the center of the channel. Calculations were performed
using the Amber force field44 within the HyperChem molecular
modeling package. Before calculations could begin, the Pap residue
had to be parametrized for use with the force field. Separate parameters
were developed for cis and trans conformations of Pap. Equilibrium
bond lengths and angles were taken from the corresponding X-ray
structures.45,46 Atom-centered partial charges were calculated using ab
initio methods on the model compound N-acetyl-N′-methyl-phenylazo-
phenylalanine carboxamide. This compound was built in Spartan, and
ø3 and ø4 torsion angles (Figure 1) were adjusted to either (a) +57°,
+57°, (b) -57°, -57°, (c) +123°, +123°, or (d) -123°, -123°, to
produce the four expected low-energy conformers of cis-Pap, each of
which has a different set of partial charges. These angles (ø3 and ø4)
were set to 0° for trans-Pap. The angles ø1 and ø2 were set at -60°
and +100° throughout. Single-point energy Hartree-Fock ab initio
calculations were performed on each of these molecules using the
6-31G* basis set. Partial charges were then assigned using the
electrostatic potential fitting algorithm within Spartan.
Conformational energy as a function of ø1 and ø2 was calculated
using HyperChem (with the Amber force field) for each of the four
different cis-Pap conformations. The angles ø1 and ø2 were each rotated
through 360° in 4-deg increments, and single-point energies were
calculated. A constant dielectric (ꢀ ) 5) was used with no nonbonded
cutoffs, and 1-4 scale factors were set at 0.5. Electrostatic interaction
energies between the Pap partial charges and the Na+ ion were
calculated for each conformation by removing the rest of the molecule
and setting the partial charges of backbone and â-CH2 group atoms of
the Pap residue to zero. Side-chain dipole moments were calculated
and represented in Grasp (v.1.2)47 on the basis of the assigned partial
charges.
Results
The gramicidin analogue Pap1-gA (Figure 1) was synthesized
via replacement of Val1 in natural gramicidin A using a
modification of the procedure described by Weiss and Koeppe.40
Figure 2 shows UV-vis spectra of Pap1-gA in methanol in dark-
adapted (trans) and photoirradiated (predominantly cis21) states.
As expected,22 the azobenzene π-π* band (330 nm) decreased
in intensity upon irradiation, the n-π* band (430 nm) increased,
and the Trp absorption (280, 290 nm) was essentially unaffected.
The current through lipid bilayers containing the gramicidin deriva-
tives was measured, and the voltage was set using an Axopatch 1D
patch-clamp amplifier (Axon Instruments) controlled by Synapse
(Synergistic Research Systems) software. Single-channel events were
recorded for a period of several hours for each set of experimental
conditions. Data were filtered at 50 or 100 Hz, sampled at 1 kHz, stored
directly to disk, and analyzed using Synapse and Igor (Wavemetrics,
Inc.) software. Mean lifetimes and current amplitudes were determined
by manual fitting of appropriate functions to the corresponding
histograms using the program Mac-Tac (Version 2.0, Instrutech Corp.).
Molecular Modeling. All molecular modeling was performed on a
Silicon Graphics Octane system (Irix 6.4) using Spartan (SGI version
5.0.3) and HyperChem (SGI version 4.5) software packages.
(44) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.;
Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179-5197.
(45) Hampson, G. C.; Robertson, J. M. J. Chem. Soc. 1941, 409-413.
(46) Brown, C. J. Acta Crystallogr. 1966, 21, 146-152.
(43) Busath, D.; Szabo, G. Biophys. J. 1988, 53, 689-695.
(47) Nicholls, A.; Sharp, K. A.; Honig, B. Proteins 1991, 11, 281-296.

Optical Switching of Ion-Dipole Interactions
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6367
Figure 4. Single-channel amplitude histograms and representative
single-channel events (inset) of dark-adapted (trans) Pap1-gA (A),
photoirradiated Pap1-gA (a mixture of Pap1-gA(cis/cis) and Pap1-gA-
(cis/trans;trans/cis channels) (B), and native gA (C). The currents were
obtained at +200 mV, 1.0 M CsCl, 5 mM BES, pH 7, DPhPC/decane
membrane; filtering was at 100 Hz. Several hundred channels of each
type were characterized.
Figure 3. Single-channel current amplitude histograms and representa-
tive single-channel events (inset) of dark-adapted (trans) Pap1-gA (A),
extensively photoirradiated Pap1-gA (predominantly Pap1-gA(cis/cis))
(B), and native gA (C). The currents were obtained at +200 mV, 1.0
M NaCl, 5 mM BES, pH 7, DPhPC/decane membranes; filtering was
at 100 Hz. Several hundred channels of each type were characterized.
Table 1. Functional Properties of Pap1-gA Channelsa
1.3 pA level to a superposition of the trans/cis and cis/trans
channels (vide infra). The changes in conductance of Pap1-
gramicidin channels seen upon photoirradiation at 337 are
reversible upon exposure to broad spectrum longer-wavelength
light; i.e., one observes a decrease in the frequency of appear-
ance of cis/cis channels with a consequent increase in the
occurrence of cis/trans, trans/cis, and trans/trans channels. The
lifetimes of all these states are similar to one another as well as
to those of native gA channels (Table 1).
1 M NaCl
1 M CsCl
0.1 N HCl
channel type
Λ
τ
Λ
τ
Λ
τ
gA
13
8
6.5
6
1.18 46
0.30 34
31.5
0.22 29
0.43 170 0.17
0.23 160 0.06
Pap1-gA(trans/trans)
Pap1-gA(cis/trans)
Pap1-gA(cis/cis)
0.11 172 0.06
0.27
0.23
hybrid gA/Pap1-gA(trans)
hybrid gA/Pap1-gA(cis)
40
38
Figure 4 shows single-channel recordings of Pap1-gA with 1
M CsCl as the electrolyte and an applied voltage of 200 mV. A
pattern is observed similar to that with the NaCl solution except
that the differences in conductance between the states are smaller
fractions of the total conductance.
Single-Channel Current-Voltage (I-V) Relationships.
The shape of the single-channel current-voltage (I-V) relation-
ship depends on the potential profile experienced by ions as
they cross the membrane field.26,48 At high permeant ion
concentrations (e.g., 1 M NaCl or 1 M CsCl), with diphytanoyl-
phosphatidylcholine membranes, ion translocation through the
pore is believed to be rate limiting.28,49-51 If a modification of
the channel at the center of the membrane were to increase the
barrier for translocation, an increase in the superlinearity of the
I-V curve would be expected to result.37
a Λ, single-channel conductance (pS) measured at 200 mV; τ, single-
channel mean lifetime (s) measured at 200 mV.
The cis state is relatively stable; it reverts to the trans state with
a half-life (in the dark) of >10 h at room temperature in
methanol or saline solution (pH 7.0) and >2 h in 0.1 N HCl.
Effects of Photoirradiation on Single-Channel Currents.
Single-channel recordings of Pap1-gA with 1 M NaCl as the
electrolyte and an applied potential of 200 mV are shown in
Figure 3. Dark-adapted Pap1-gA channels (Figure 3A) exhibit
about 60% of the conductance of native gA (Figure 3C) under
these conditions. The mean lifetimes are similar; the lifetime
of Pap1-gA(trans) is 0.3 s, whereas that of native gA is 1.2 s
(Table 1). Irradiation of dark-adapted Pap1-gA leads to the
formation of Pap1(cis)-gA (Figure 3B). The presence of both
Pap1(trans)-gA and Pap1(cis)-gA in the membrane can, in
principle, lead to conducting dimers of four types: trans/trans,
cis/trans, trans/cis, and cis/cis. In the present case, three different
levels of current were distinguished: 1.2, 1.3, and 1.6 pA. The
1.6 pA level corresponds to the dark-adapted, and therefore,
the trans/trans state. The relative abundance of the 1.2 and 1.3
pA levels was dependent on the time of exposure to 337 nm
light. With extensive irradiation, only the 1.2 pA level was seen.
Therefore, we assign this level to the cis/cis channel and the
The curvature of I-V plots is best visualized by plotting
normalized conductance (i.e., the conductance measured at
voltage V divided by the conductance measured at 25 mV)
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6368 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Borisenko et al.
Figure 6. Single-channel current-voltage plots at low permeant ion
concentration (0.1 M CsCl, 5 mM BES, pH 7; filtering was at 50 or
100 Hz). Pap1-gA(cis/cis) (9), Pap1-gA(trans/trans) (b), and native gA
(2).
Figure 5. Plots of normalized conductance versus voltage. (A) 1 M
NaCl; (B) 1 M CsCl. Pap1-gA(cis/cis) (9), Pap1-gA(trans/trans) (b),
and native gA (2). All solutions contained 5 mM BES, pH 7; filtering
was at 50 or 100 Hz.
versus voltage. Figure 5 shows g(V)/g(25) versus voltage curves
observed with 1 M NaCl (Figure 5A) and 1 M CsCl (Figure
5B) as the electrolyte. With NaCl, the ratio g(V)/g(25) is greater
than or equal to 1 at all voltages; however, the values of g(V)/
g(25) are always larger for Pap1-gA(trans/trans) than for gA
and always larger for Pap1-gA(cis/cis) than for Pap1-gA(trans/
trans). That is, Pap1-gA(cis/cis) gives the most superlinear I-V
curve. The same pattern is observed with CsCl, except that at
high voltages the ratio g(V)/g(25) begins to decrease, indicating
that a process other than translocation becomes rate limiting
(since the gramicidin channel is saturable, the ratio g(V)/g(25)
must ultimately tend to zero). The voltage at which the ratio
begins to decline is noticeably higher for Pap1-gA(cis/cis) (∼300
mV) than for Pap1-gA(trans/trans) (∼200 mV) than for gA (50
mV).
At very low permeant ion concentrations and high applied
voltages, ion entry into the channel becomes rate limiting.50,52,53
Under these conditions, changes to the channel in the center of
the membrane are expected to have little effect on conductance.37
Figure 6 shows single-channel I-V curves obtained in 0.1 M
CsCl for gA, and for Pap1-gA in trans/trans and cis/cis forms.
Nonpermeant triethylammonium chloride (0.4 M) was added
to minimize interfacial polarization effects.19,53 As the voltage
is increased, all the currents tend to the same limiting value
(∼2 ( 0.2 pA) at +400 mV.
Figure 7. Current amplitude histograms of single channels obtained
with a mixture of gA and Pap1-gA(cis). (A) +200 mV applied potential.
(B) +350 mV applied potential. 1.0 M CsCl, 5 mM BES, pH 7; filtering
was at 100 Hz. Several hundred channels of each type were character-
ized.
higher than that of hybrid Pap1-gA(cis)/gA channels which, in
turn, was higher than that of Pap1-gA(trans/trans) homodimers
(Table 1).
Hybrid Channels. As a further probe of the structural and
functional effects of a valine to phenylazo-phenylalanine
substitution at position 1, we investigated the ability of Pap1-
gA peptides to form hybrid channels with native gA peptides.
Figure 7 shows current amplitude histograms obtained when
Pap1-gA(cis) and native gA were added simultaneously to
membranes with 1 M CsCl as the electrolyte. In addition to the
single-channel currents expected for Pap1-gA(cis/cis) homo-
dimers and gA homodimers, a new, intermediate level of
conductance was observed. By analogy with previous observa-
tions of hybrid channel formation with gramicidin analogues,37,54
this new level is ascribed to a hybrid channel formed by Pap1-
gA and gA monomers. Similar results were obtained when Pap1-
gA(trans) was mixed with native gA (data not shown). The
conductance of hybrid Pap1-gA(trans)/gA channels was a little
Two different hybrid channels are expected to form, i.e. Pap1-
gA/gA and gA/Pap1-gA, with opposite orientations with respect
to the applied field. These may or may not exhibit different
conductances, depending on the degree of asymmetry in the
corresponding free energy profiles for cation movement and the
size of the applied field.37,55 In the present case, no splitting of
the hybrid peak is observed at 200 mV (Figure 7A), but splitting
is observed at 350 mV (Figure 7B).
Current-voltage curves of gA/Pap1-gA(cis) hybrid channels
and gA/Pap1-gA(trans) hybrid channels in 1 M CsCl (0-400
mV range) had shapes intermediate between those of the
corresponding pure homodimers (not shown). The superlinearity
of I-V curves increased in the following order: gA/gA < gA/
Pap1-gA(trans) < gA/Pap1-gA(cis) < Pap1-gA(trans/trans) <
(52) Andersen, O. S. Biophys. J. 1983, 41, 119-133.
(53) Andersen, O. S. Biophys. J. 1983, 41, 135-146.
(54) Durkin, J. T.; Koeppe, R. E., II; Andersen, O. S. J. Mol. Biol. 1990,
211, 221-234.
(55) Andersen, O. S.; Durkin, J. T.; Koeppe, R. E., II. In Transport
through membranes: carriers, channels and pumps; Pullman, A., Jortner
J., Pullman, B., Eds.; Kluwer Academic: Dordrecht, 1988; pp 115-132.

Optical Switching of Ion-Dipole Interactions
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6369
Pap1-gA(cis/cis). Average lifetimes of gA/Pap1-gA(cis) and gA/
Pap1-gA(trans) hybrid channels were similar to those of homo-
dimer channels (Table 1).
planar, is expected to have a zero dipole moment.22 We now
consider the possible effects of the photocontrolled occurrence
of this dipole on channel activity.
Alkali cations must pass through gramicidin channels by
diffusing to the channel mouth and entering the channel, moving
through the pore (translocation), and finally, exiting from the
channel.19,51 An azobenzene group attached to position 1 of
gramicidin is located in the central part of the membrane and
would not be expected to have a significant impact on the
process of ion entry. When ion entry is made the rate-limiting
step, by having a low concentration of permeant electrolyte and
a high applied voltage, no difference is observed in the
conductance of cis and trans Pap1-gA channels (Figure 6).
Modified channels, where the modification is at the channel
entrance (e.g., gramicidin-ethylenediamine24), do not, in gen-
eral, show this behavior.
In contrast, an azobenzene dipole at the center of the
membrane might be expected to affect the translocation process.
When translocation is made rate limiting by using 1 M NaCl
or CsCl electrolyte solutions and diphytanoyl-phosphatidylcho-
line membranes,26 conductance changes are seen upon photo-
isomerization of Pap1-gA(trans) to Pap1-gA(cis) (Figures 3 and
4). In addition, the shapes of the corresponding I-V curves are
consistent with an increase in the barrier for alkali cation
permeation when the Pap group isomerizes from trans to cis
(Figure 5).
Discussion
Pap1-gA Channels Have the Same General Architecture
as Native gA Channels. Before considering the mechanism
whereby photoisomerization of Pap1-gA alters channel function,
one must first determine whether a substantial change in the
overall structure of the channel has occurred as a result of the
introduction of the Pap group. A stringent test of the degree to
which the channel structure has been altered by chemical
modification is the ability of modified peptides to form
heterodimers with native gA peptides.54 If the structure of Pap1-
gA is exactly like that of gA, then one would expect no
difference between the interaction energies of gA homodimers
and gA/Pap1-gA heterodimers. If there is no energetic difference,
the following relationship should hold:37,54,56
φΗ/2(φΑφΒ)1/2 g 1
(1)
where φH is the frequency of appearance of hybrid channels
(of either orientation), and φΑ and φΒ are the frequencies of
appearance of gA and Pap1-gA homodimer channels. When gA/
Pap1-gA(cis) hybrid channels were examined (Figure 7), the
ratio φΗ/2(φΑφΒ)1/2 was found to be 1.12 at 200 mV. For hybrid
gA/Pap1-gA(trans) channels, the ratio was 1.02. Both of these
ratios are very close to 1, indicating that there is very little
energetic difference between homo- and heterodimer channels.
This result is strong evidence that the overall structure of the
Pap1-gA channel is the same as the native gA channel, that is,
a â-helical dimer.57
The effect of photoisomerization is more pronounced with
NaCl than CsCl (Figures 3-5). This presumably reflects
differences in the detailed free energy profiles for these ions; a
similar pattern has been seen with other position 1 mutations
that alter the dipole moment of the side chain.36 Indeed, proton
flux through the channel, which is expected to be governed by
an entirely different free energy profile,29,58-60 was found to be
almost unaffected by photoisomerization (Table 1).
Trans-to-Cis Photoisomerization of Pap1-gA Increases the
Barrier for Alkali Cation Translocation. Since we are
primarily interested in the effects of photoisomerization, we will
focus on the conductance differences between Pap1-gA(trans)
and Pap1-gA(cis) channels. However, we note there is also a
difference in the conductance properties of Pap1-gA(trans) and
native gA channels. Koeppe and Andersen and colleagues36,56
have reported the channel properties of position 1 mutants in
which valine was altered to phenylalanine and a series of
substituted phenylalanine derivatives. If one considers the
phenylazo group as a substituent of phenylalanine, the observed
conductance of Pap1-gA(trans/trans) (1 M NaCl) is consistent
with the observed trends reported by Koeppe et al.36,56 The low-
voltage conductance of Pap1-gA(trans/trans) is 7 pS vs 10.2 pS
for Phe1-gA, 6 pS for p-F-Phe1-gA, 3.9 pS for p-OH-gA, and
12.5 pS for gA (1 M NaCl).36,56 Most of these conductances
could be rationalized by considering the projections of group
moments of the side chains along the R-â bond. A similar
analysis for Pap1-gA(trans/trans) would presumably explain why
the conductance of Pap1-gA(trans/trans) falls in the middle of
the observed range. However, since we were primarily interested
in comparing cis and trans Pap1-gA channels to one another,
rather than to native gA, we chose to represent side-chain dipole
moments using atom-centered partial charges as this proved
more amenable for use with the molecular mechanics algorithms
used for conformational analysis (vide infra).
Molecular Origins of the Effects of Photoisomerization
on Conductance. If the photocontrolled introduction of a dipole
at the center of the membrane is, indeed, responsible for the
observed effects on alkali cation conductance, then the dipole
must act in such a way as to increase the barrier for ion
translocation. Taking the native gramicidin channel structure
as determined by solid-state NMR as a starting point,57 we
performed energy calculations to identify conformations that
would be available to a cis-Pap group at position 1 in the
channel. We assumed that the ø3 and ø4 torsion angles (Figure
1) of the Pap group would be close to those observed in the
azobenzene crystal.45 Four pairs of ø3, ø4 torsion angles are
thus possible (enantiomeric forms of an isolated azobenzene
become diastereomeric when attached to gramicidin). For each
pair of ø3, ø4 torsion angles, a systematic calculation of
conformational energy versus the torsion angles ø1 and ø2 was
carried out assuming the conformation of the rest of the molecule
was unchanged. This simple search identified relatively restricted
regions of the ø1, ø2 map which were energetically allowed
(Figure 8). These regions were determined almost entirely by
van der Waals interactions, i.e. steric clashes, with other side
chains.
For each value of ø1 and ø2, we also calculated the
electrostatic energy of interaction between the Pap dipole
(represented by atom-centered partial charges) and a sodium
An isolated cis-azobenzene group with ø3 and ø4 torsion
angles near 55° (as observed in the crystal45) has a dipole
moment of approximately 3.0 D; the trans conformation, if
(58) Quigley, E. P.; Quigley, P.; Crumrine, D. S.; Cukierman, S. Biophys.
J. 1999, 77, 2479-2491.
(56) Mazet, J. L.; Andersen, O. S.; Koeppe, R. E., II. Biophys. J. 1984,
45, 263-276.
(57) Kovacs, F.; Quine, J.; Cross, T. A. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 7910-7915.
(59) Sadeghi, R. R.; Cheng, H. P. J. Chem. Phys. 1999, 111, 2086-
2094.
(60) Schumaker, M. F.; Pomes, R.; Roux, B. Biophys. J. 1999, 76, A442-
A442.

6370 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Borisenko et al.
Figure 9. (A) Top view and (B) side view of Pap1-gA(cis/cis)
gramicidin. One low-energy conformer (ø1 ) 180.0°, ø2 ) -90.0°,
ø3 ) 56.9°, ø4 ) 56.9°) of Pap1 is shown. The azobenzene side-chain
dipole of one Pap group is represented as a vector (arrowhead at the
positive end) projecting into the channel lumen (drawn at a scale of
0.06 D/Å with the origin of the vector at the center of the actual dipole).
(C) Top view and (D) side view of Pap1-gA(trans/trans) (ø1 ) 180.0°,
ø2 ) -90.0°, ø3 ) 0°, ø4 ) 0°).
Figure 8. Conformational energy contours (dark lines) and electrostatic
energies (shaded zones) as a function of torsion angles ø1 and ø2 in
Pap1-gA(cis/cis) for the four low-energy ø3, ø4 conformers of cis-
Pap: (A) ø3, ø4 ) -56.9°, (B) ø3, ø4 ) 123.1°, (C) ø3, ø4 ) 56.9°,
(D) ø3, ø4 ) -123.1°. Conformational energy is lowest (∼0 kcal/
mol) for ø1 ≈ -90 and ø2 ≈ +90 (A,D) or -90 (B,C). Contours are
drawn at 10 kcal/mol intervals; conformational energy is thus prohibi-
tively high throughout most of the ø1, ø2 map. The scale for the
electrostatic interaction energies (shaded zones) between Pap partial
charges and a sodium ion at the center of the channel is given at the
bottom of the figure.
of Pap1-gA(trans/trans) to Pap1-gA(cis/cis) would result in an
increase in the central barrier by about 0.4-3 kcal/mol (0.2-
1.5 kcal/mol for each Pap). The observed decrease in conduc-
tance (Figure 3, Table 1) indicates a change in barrier height of
about 0.2 kcal/mol (for Na+), close to the low end of this
calculated range. Thus, photoswitching of ion dipole interactions
can, semiquantitatively, account for the observed effects on
conductance. The overestimation of the electrostatic contribution
may reflect the simplicity of the model (e.g., the fact that the
discontinuous dielectric is neglected), or that other molecular
details play a role. For instance, lipid packing might be expected
to be significantly different around the cis and trans conforma-
tions (Figure 9).
ion positioned at the center of the channel. Since we were
primarily interested in whether the electrostatic interaction was
favorable or unfavorable, we used a uniform dielectric constant,
ꢀ ) 5, reported by Hu and Cross33 to correctly reflect the effects
of Trp dipoles on gramicidin conductance, measured under
conditions similar to those used here. A more sophisticated
treatment would consider the influence of the dielectric bound-
aries of the system28,61 and the dynamics of the side chains on
the magnitude of the interaction energy.62 Nevertheless, we
found that throughout the conformationally allowed zones, and
indeed for all Values of ø1 and ø2, the electrostatic energy of
interaction was positive and varied between 0.2 and 1.5 kcal/
mol (Figure 8), a value similar in magnitude, but opposite in
sign, to those estimated by others for the energy of interaction
between Trp dipoles and a cation in the channel.28
If we assume that the electrostatic energy of interaction
between a trans-Pap group (which has almost no dipole) and a
sodium ion in the channel is zero, this conformational analysis
indicates that a cis-Pap group would always be expected to
increase the barrier to alkali cation movement via an electrostatic
mechanism since the positive end of the side-chain dipole is
always oriented toward the center of the channel (Figure 9).
Since the two Pap groups in a (dimer) channel are approximately
on opposite sides of the channel (Figure 9), we assume that
their interactions with ions in the pore are independent and
additive. The finding that the conductances of heterodimer
channels (which contain one Pap group) are intermediate
between native gramicidin channels and homodimer Pap chan-
nels supports this assumption, i.e., that the two Pap groups act
independently and additively. Given these assumptions, this
simple electrostatic analysis suggests that photoisomerization
With the present configuration, the sizes of the effects of
photoisomerization are probably not large enough to permit
Pap1-gA to be of use for manipulating cellular excitability. The
photomodulated properties of Pap1-gA may be of use, however,
for resolving gramicidin-associated currents from background
currents in the gramicidin-based biosensor described by Cornell
and colleagues.63,64 The predictability of the system does suggest
that larger dipole changes or changes closer to the central axis
of the pore could effect substantially larger changes in conduc-
tance. Compounds that undergo larger dipole moment changes
upon photoisomerization such as spiropyran/merocyanine-based
compounds could be substituted for azobenzene in the channel.
Strategies for increasing the effects of photoisomerization on
channel conductance might also include the use of conforma-
tionally restrained azobenzene analogues to control the orienta-
tion of the side-chain dipoles.65
Acknowledgment. We thank NSERC Canada for financial
support.
JA000736W
(63) Woodhouse, G.; King, L.; Wieczorek, L.; Osman, P.; Cornell, B.
J. Mol. Recognit. 1999, 12, 328-334.
(64) Cornell, B. A.; Braach-Maksvytis, V. L.; King, L. G.; Osman, P.
D.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-583.
(65) Zhang, J.; James, D. A.; Woolley, G. A. J. Pept. Res. 1999, 53,
560-568.
(61) Martinez, G.; Sancho, M. Eur. Biophys. J. 1993, 22, 301-307.
(62) Woolf, T. B.; Roux, B. Biophys. J. 1997, 72, 1930-1945.