Functional Dynamics of Ion Channels: Modulation of Proton
Movement by Conformational Switches
Ching-Hsing Yu and Re´gis Pome`s*
Contribution from the Structural Biology and Biochemistry Program, Hospital for Sick Children,
and Department of Biochemistry, UniVersity of Toronto, Toronto, Ontario, Canada
Received March 25, 2003; E-mail: pomes@sickkids.ca
Abstract: Detailed comparative studies of proton relay in native and chemically modified gramicidin channels
provide a unique opportunity to uncover the structural basis of biological proton transport. The function of
ion channels hinges on their ability to provide surrogate solvation in narrow pore filters so as to overcome
the dielectric barrier presented by biological membranes. In the potassium channel KcsA and in the cation
channel gramicidin, permeant selectivity and mobility are determined by the proteinaceous matrix via
hydrogen bonding, charge-dipole, and dipole-dipole interactions. In particular, main-chain carbonyl groups
in these pore interiors play an essential role in the solvation of alkali ions and of protons. In this study,
molecular dynamics simulations reveal how the translocation of H+ is controlled by nanosecond
conformational transitions exchanging distorted states of the peptidic backbone in the single-file region of
a dioxolane-linked analogue of the gramicidin dimer. These results underline the functional role of channel
dynamics and provide a mechanism for the modulation of proton currents by fluctuating dipoles.
Introduction
Understanding the molecular determinants of proton move-
ment in membrane proteins is necessary in order to elucidate
biological energy conversion.1 In Grotthuss relay mechanisms,2
the rapid long-range transport of protons is mediated by small
fluctuations in the arrangement of relay groups forming
hydrogen-bonded networks.3 Heavy-atom displacements of the
order of 0.01-0.1 nm result in H+ translocation over distances
of 1 nm in nanosecond time scales. This process of structural
diffusion is beginning to be understood in bulk water4,5 and in
the array of water molecules (proton wires) embedded in the
gramicidin channel.6,7
Gramicidin is a pentadecapeptide that assembles as a non-
covalent head-to-head dimer in lipid bilayers to form a channel
permeable to small monovalent cations.8 Its main chain adopts
a right-handed â-helical structure resulting in a narrow cylindri-
cal pore lined with peptide bonds.9,10 The lumen accommodates
a single file of up to eight water molecules (water wire) that
mediates the passive conduction of protons via a hop-and-turn
Grotthuss mechanism.11,12 Structural fluctuations of the hydrogen-
bonded network involving water molecules and the channel
backbone give rise to the translocation of an ionic defect
(movement of the excess proton via successive transfers or hops
of H nuclei) and of a bonding or turning defect (reorientation
of the water chain) restoring the polarization of the wire prior
to the passage of another proton in the same direction.7 The
local environment primes the wire for both hop and turn steps.
Hydrogen-bond donation by each water molecule in the wire
to carbonyl O atoms of the channel helps solvate both
elementary forms of the hydrated proton, namely, the hydronium
(OH3+) and Zundel (O2H5+) cations, and catalyzes the reorga-
nization of the network.6
Chemical modifications of gramicidin A (gA) offer a unique
avenue to refine our understanding of structural diffusion at the
molecular level. The insertion of the five-member ring dioxolane
between the two formylated N-termini of gramicidin results in
covalently linked dimers that form channels.13 The two dias-
tereoisomeric forms of the linked dimers, respectively, SS and
RR, differ in their proton permeation properties.14 The proton
conductance, gH, of the RR channel is reduced by a factor of 2
to 4 compared to that of native and SS dimers. Detailed atomic
models of the linked channels suggest that functional differences
are a consequence of structural distortions in the middle of the
channel.15 Here, we investigate the physical basis for the
modulation of proton currents through a comparative study of
the hop-and-turn mechanism in native, SS, and RR dimers. To
this end, we use molecular dynamics simulations extending to
several tens of nanoseconds (see Methods). Because this time
scale well exceeds that of structural diffusion in the lumen,7
(1) Saraste, M. Science 1999, 283, 1488.
(2) Grotthuss, C. J. T. de. Ann. Chim. 1806, 58, 54.
(3) Nagle, J. F.; Morowitz, H. J. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 298.
(4) Agmon, N. Chem. Phys. Lett. 1995, 244, 456.
(5) Tuckerman, M.; Laasonen, K.; Sprik, M.; Parrinello, M. J. Phys. Chem.
1995, 99, 5749.
(6) Pome`s, R.; Roux, B. Biophys. J. 2002, 82, 2304.
(7) Pome`s, R.; Roux, B. Biophys. J. 1996, 71, 19.
(8) Tian, F.; Cross, T. A. J. Mol. Biol. 1999, 285, 1993.
(9) Arseniev, A. S.; Barsukov, I. L.; Bystrov, V. F.; Lomize, A. L.;
Ovchinnikov, Y. A. FEBS Lett. 1985, 186, 168.
(10) Ketchem, R. R.; Hu, W.; Cross, T. A. Science 1993, 261, 1457.
(11) Akeson, M.; Deamer, D. W. Biophys. J. 1991, 60, 101.
(12) Levitt, D. G.; Elias, S. R.; Hautman, J. M. Biochim. Biophys. Acta 1978,
512, 436.
(13) Stankovic, C. J.; Heinemann, S. H.; Delfino, J. M.; Sigworth, F. J.;
Schreiber, S. L. Science 1989, 244, 813.
(14) Quigley, E. P.; Quigley, P.; Crumrine, D. S.; Cukierman, S. Biophys. J.
1999, 77, 2479.
(15) Yu, C.-H.; Cukierman, S.; Pome`s, R. Biophys. J. 2003, 84, 816.
Published on Web 10/18/2003
13890 9 J. AM. CHEM. SOC. 2003, 125, 13890-13894 10.1021/ja0353208 CCC: $25.00 © 2003 American Chemical Society

this study provides unprecedented insight into the functional
role of channel dynamics. Free energy profiles characterize the
interplay between structural fluctuations of the water-filled
channels and the translocation of a bonding defect and of an
excess proton in the water wire.
Methods
The molecular system consisted of the dimers together with eight
water molecules in the single file region, two at the interface, and
cylindrical caps of water molecules at each mouth of the channel as in
earlier studies.6 New parameters for the dioxolane linkers based on ab
initio calculations were developed15 and combined with the CHARMM22
force field16 used for the polypeptide. The conventional TIP3P water
model17 was used to represent the wire in the turn step, whereas the
PM6 model,18,19 a polarizable and dissociable empirical force field
consisting of O2- and H+ moieties, was used as before in both turn
and hop steps.6 Matching caps of 14 water molecules were used for
the turn step, whereas caps of 36 TIP3P water molecules were employed
for the hop step. No cutoff was employed in the calculation of
nonbonded interactions, and a dielectric constant of 1 was used
throughout. A previous study of proton translocation in native gA
suggested that caps of 36 water molecules are sufficiently large to
capture the primary influence of outlying water on the distribution of
the excess proton in the single-file region.6 As described elsewhere,6
artificial restraints were applied to the eight Trp indole rings of the
channel, to prevent unfolding in the absence of the lipid bilayer, and
to water molecules, to prevent diffusion and evaporation. Despite the
neglect of the lipid bilayer and the use of artificial restraints, the average
tilts of peptide planes and their dynamic fluctuations were shown in
an earlier study of the dimers in vacuo15 to be commensurate with
experimental data obtained in NMR studies of native gA in lipid. Results
reported below for turn and hop steps in the native gA dimer were
obtained, respectively, from a previous study6 and from its extension
by 6 ns. The initial structures of the SS and RR dioxolane-linked dimers
were taken from a previous study of the empty channels.15
Langevin integration with a friction coefficient of 5 ps-1 imposed
on heavy atoms was propagated at 300 K with the CHARMM program20
to generate molecular dynamics trajectories for each of the two linked
channels, successively without and with an excess proton. Each
simulation of the unprotonated linked dimers with the TIP3P water
model was carried out with an integration time step of 2 fs and extended
to a total of at least 20 ns of equilibration and 36 ns of production. All
the simulations with the PM6 model used a time step of 1 fs. They
extended to 12 and to at least 36 ns for the unprotonated SS and RR
dimers, respectively. In the latter case, umbrella simulations were
employed to improve the sampling efficiency along íz, the axial
component of the total dipole moment of the water wire. Biasing
potentials of the form U(íz) ) 1/2kí(íz - íz°)2 were imposed, with a
force constant kí ) 2.0 kcal/mol e-2 Å-2 and íz° ranging from 0 to 9
e Å in increments of either 0.5 or 1 e Å. A set of umbrella calculations
was also performed for each of the four conformers 1 to 4 with
additional harmonic restraints on the RR linker’s backbone torsions ı
and ı′ (see ref 15) with a force constant of 3.28 kcal/mol/rad2 and
reference (ı,ı′)0 ) (-105.0,-18.5), (-36.0, -36.0), (-18.5,-105.0),
and (-108.0, -108.0), respectively, for conformers 1, 2, 3, and 4. The
system was reequilibrated as before7 following the addition of an excess
proton between two water molecules near z ) 0, and hydrogen nuclei
were subsequently free to move in the single-file chain without any
bias on proton position or channel backbone. Simulations of the
protonated wire in the linked dimers comprised 36 and 60 consecutive
1 ns trajectories, respectively, for the SS and RR channels.
Results
Both SS and RR linked channels conserve the overall
secondary structure of native gA, with peptide planes nearly
aligned with the helical axis (Figure 1B).15 In all three channels,
most peptide planes undergo rapid (picosecond to nanosecond)
librations about the axis defined by the two adjacent CR atoms,
with an rms amplitude of (8° to (15° on average. Such
plasticity of the channel backbone, which is commensurate with
experimental data,15 is thought to play a functional role: when
they tilt toward the inside of the channel lumen, carbonyl groups
help solvate alkali ions permeating through the pore via charge-
dipole interactions.8,21 In native and SS dimers, librations of
the two N-terminal peptide planes, like those of all other peptide
planes, are unimodal and centered near zero, which corresponds
to alignment with the axis of the channel (Figure 1A,B). The
only significant difference presented by the RR channel lies in
the structure and fluctuations of the backbone next to the
linker.15 Distortions due to the chirality of the RR linker force
the two peptide planes flanking the dioxolane ring to tilt out of
alignment with the helix axis by 30° on average. Thermally
activated transitions exchange the topology of each of the two
central CO groups with respect to the channel lumen. Four
conformers henceforth labeled 1 through 4 are characterized
by these two carbonyl groups pointing (in, out), (out, out), (out,
in), and (in, in), respectively (Figure 1C). In the absence of an
excess proton, conformational switching across free energy
activation barriers of 2.1 to 2.8 kcal/mol results in an equilibrium
between the four RR conformers on a nanosecond time scale.
Addition of an excess proton to the water wire displaces the
equilibrium distribution by destabilizing (out, out) conforma-
tions. A similar effect is also visible in native and SS dimers.
The relative preference for asymmetric conformers 1 and 3 is
exacerbated by the presence of H+.
The reorientation of water molecules results in the translo-
cation of a bonding defect or turn step of the Grotthuss
mechanism.3,7 Comparison of potential of mean-force (PMF)
profiles to invert the polarity of the unprotonated wire (Figure
2A) shows that the polarization of water molecules in the middle
of the pore is highly sensitive to the topology of carbonyl groups
at the dimer junction. This effect is highly consistent for both
water models used in the study. Although the wire is polarized
in native gA,6 there is no such preference in the SS dimer, and
each conformer of the RR channel gives rise to a different
profile. The reversible thermodynamic work required to reorient
the water wire is determined by the topology of the CO groups
adjacent to the RR dioxolane ring. When a CO points out, a
substantial activation energy barrier (similar to that of native
gA) opposes the reorientation of the three innermost water
molecules in the same monomer moiety, whereas when the CO
points in, the process is activationless (as in the SS dimer)
(Figure 2B). These results underline the role of dipole-dipole
as well as hydrogen-bonding interactions between water mol-
ecules and the channel in the migration of a bonding defect.
(16) MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck,
J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy,
D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.;
Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich,
M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera,
J.; Yin, D.; Karplus, M. J. Phys. Chem. B 1998, 102, 3586.
(17) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein,
M. L. J. Chem. Phys. 1983, 79, 926.
(18) Stillinger, F. H.; David, C. W. J. Chem. Phys. 1978, 69, 1473.
(19) Weber, T. A.; Stillinger, F. H. J. Phys. Chem. 1982, 86, 1314.
(20) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan,
S.; Karplus, M. J. Comput. Chem. 1983, 4, 187. (21) Roux, B.; Karplus, M. J. Phys. Chem. 1991, 95, 4856.
Functional Dynamics of Ion Channels A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 125, NO. 45, 2003 13891

These results also suggest that the activation energy barrier for
water reorientation in native gA is due to the significant
preference for an outward tilt of both N-terminal CO groups in
the absence of H+ (see Figure 1A).
Backbone distortions of the RR linked channel modulate the
equilibrium distribution of the excess proton (Figure 3). The
effect of linkage depends on the chirality of the dioxolane ring
(Figure 3A). The relative proton affinity of the eight single-file
water molecules in the SS channel is essentially identical to
that in native gA, with a broad distribution peaking near the
dimeric interface (water molecules #5 and #6). By contrast, the
average proton distribution peaks in each monomer moiety of
the RR channel. The mean position of the excess proton strongly
depends on the conformation of the RR linker. In each of the
asymmetric conformers 1 and 3, H+ is preferentially hosted in
the monomer in which one of the two CO groups tilts into the
lumen, by or near a water molecule aligned with the amide
dipole moment (Figure 3A,B). In confomer 4, where both CO
groups point in, the sum of the two amide dipole moments is
aligned with the dimer interface and the excess proton is
preferentially shared by the two central water molecules in a
Zundel cation.
The nature of the coupling between proton movement and
conformational isomerization of the RR linker is revealed by
the two-dimensional free energy surface correlating the position
of the excess proton to the linker conformation (Figure 3C).
Each of the three channel conformations 4, 3, and 1 corresponds
to a trough in the free energy surface. The fact that the position
of the two ridges separating these three valleys is essentially
independent of the proton position (i.e., they are aligned
with the ordinate axis) indicates that the isomerization of the
channel backbone controls the progress of the ionic defect, not
the other way around. The movement of the excess proton from
one end of the single-file region to the other requires confor-
mational isomerization of the linker, whereas conformational
isomerization does not require proton movement. Thus, an
excess proton starting from the upper part of the channel (top
right corner region of Figure 3C) dwells in the top monomer in
conformation 1, with the top CO pointing in; following an
activated transition flipping the bottom CO from out to in
(activation free energy of 1.8 ( 0.2 kcal/mol), the proton
becomes localized in the middle of the channel with both COs
pointing in (conformer 4). This conformation constitutes an
intermediate state in the preferred translocation pathway. From
there, the channel backbone rapidly decays either back to 1 or
on to 3, following the flip of the top CO from in to out. In the
latter case, the excess proton completes its journey past the
middle turn of the helix and on to the lower monomer (bottom
left corner).
Discussion
These results highlight the role of backbone carbonyl groups
and of nanosecond fluctuations of the polar environment in
proton transport mechanisms. It was recognized early that rapid
Figure 1. (A) Potential of mean force for backbone fluctuations, respectively, (top) in native gA and (middle) in SS and (bottom) RR linked dimers. The
orientation of the two N-terminal peptide planes is characterized by the tilt angles R and R′, which are defined as zero when the mean plane of the amide
group is collinear with the channel axis and positive when the carbonyl group points into the lumen. Left: with water only. Right: with water and an excess
proton in the channel lumen. Contour spacing is 0.5 kcal/mol. The small deviation from symmetry with respect to the R ) R′ diagonal suggests adequate
statistical convergence. (B) Representative snapshot of the backbone of the RR dimer emphasizing the linker and its two flanking CO groups, which are
displayed with thicker bonds. The two carbonyl groups bonded to the dioxolane ring in the top and bottom gramicidin monomers are, respectively, in and
out of the lumen (conformer 1). (C) Representative snapshots of the RR linker in this and subsequent figures highlighting the topology of carbonyl groups
in conformers 1 through 4. Structural representations in this and subsequent figures were prepared with the program VMD.32
A R T I C L E S Yu and Pome`s
13892 J. AM. CHEM. SOC. 9 VOL. 125, NO. 45, 2003

(picosecond) librations of the peptidic backbone play a role in
the mobility of the lumen contents22 and that a moderate inward
tilt of carbonyl groups provides stabilization of alkali cations
so as to make up for their partial desolvation in the narrow pore
interior.8 All the peptide carbonyl groups in the native and the
SS-linked channels, and all but two of them in the RR channel,
undergo such oscillations freely with an average tilt of 0° to
(20°.15 The only significant difference between the three
channels is that, in the RR linker, the tilt of two peptide planes
is more pronounced and presents a bimodal character, with
thermally activated transitions governing the exchange between
inward- and outward-facing conformations of the carbonyl
groups. Together, these two peptides appear to act as “push
switches” that control the passage of protons.
(22) Chiu, S. W.; Jakobsson, E.; Subramaniam, S.; McCammon, J. A. Biophys.
J. 1991, 60, 273.
Figure 2. (A) Potential of mean-force profiles for the reorientation of water
molecules in gA, SS, and RR channels and in RR conformers 1 through 4.
íz is the projection of the total molecular dipole of the single-file water
chain along the channel axis. Results obtained with the PM6 and TIP3P
water models are shown in solid and dashed lines, respectively. In the latter
case, íz was scaled by 0.417, the partial charge of H atoms in the TIP3P
model, for direct comparison with the PM6 model, in which the formal
charge of H atoms is 1. The two water models yield qualitatively consistent
profiles. Local minima and inflection points mark the progression of the
bonding defect among the 10 water molecules from mouth to mouth. (B)
Representative conformations of linked channels and their lumen contents.
(Top) Half-oriented water chain in the SS channel; the bonding defect is
on the fourth water molecule from the top, which donates a hydrogen to
one of the two CO groups adjacent to the linker. (Bottom) Polarized water
chain in conformer 2 of the RR channel; in that snapshot, the bonding defect
is on the ninth water molecule.
Figure 3. (A) Equilibrium distribution of the excess proton among the
eight innermost single-file water molecules (indices 2 to 9) in native gA,
in SS and RR channels, the latter of which is also shown broken down into
RR conformers 3, 4, and 1. The O atom hosting H+ at any given time is
determined as that for which the distance to the third-nearest H nucleus is
shortest among the single-file water molecules. Escape of the excess proton
from the channel was precluded by construction.6 (B) Representative
conformations of RR conformers 3, 4, and 1, respectively, with H+ hosted
by water #7 as a hydronium ion, shared by waters #5 and #6 as a Zundel
cation, and hosted by water #4 as hydronium. Note how the location of the
excess charge is aligned with the dipole moment of the CO group(s) tilted
into the lumen. (C) Potential of mean force for the position zH+ of an excess
proton along the axis of the RR channel as a function of channel
conformation. In the symmetric conformation 4, the difference between the
tilt angles of the two CO groups, R - R′, is near zero, whereas, in the two
asymmetric conformations 1 and 3, the difference is, respectively, large
and positive and large and negative. Contour spacing is 0.5 kcal/mol. The
lowest free energy pathway for the translocation of H+ from end to end of
the single file involves successive conformational switches exchanging the
topology of the two central CO groups.
Functional Dynamics of Ion Channels A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 125, NO. 45, 2003 13893

By comparing the molecular mechanism in highly analogous
systems, we are able to ascribe experimentally measured
functional differences to the structural and dynamic properties
of the channel. While conformational switching modulates both
hop and turn steps of the Grotthuss mechanism, its effect on
the turn step is unlikely to control the overall rate of proton
permeation through the pore. On one hand, proton conductance
of the SS dimer is similar to that of native gA14 despite the
dramatic drop in the height of the activation free energy barrier
opposing water reorientation (Figure 2). On the other hand, the
proton distribution is nearly identical in native and SS channels
but differs in the RR dimer. The asymmetric conformers of the
RR linker are unique in their ability to promote the localization
of H+ in one monomer through charge-dipole interactions,
suggesting a mechanism for the modulation of proton conduc-
tion, whereby activated switching of one of the two central
peptides from outward to inward tilt limits the progress of the
excess proton.
This work opens the way to comparative studies of structure-
function relationships in proton ducts. More remains to be done
to confront the results presented here to experimental conduc-
tance data using a detailed kinetic model.23 It has been proposed
that the exit of protons from the lumen is the rate-limiting step
for proton transport in gA.24 The present study focuses on the
single-file region of the channel and does not exclude that the
rate of proton entrance and/or exit could be modified indirectly
by RR linkage, either through a change in the proton affinity
of the channel relative to that of the bulk solution or through
modulation of collective motions of the channel in its lipid
environment. The effect of linkage on the transport properties
of gA channels embedded in a lipid bilayer will be the object
of further studies. Nevertheless, the above results afford
unprecedented insight into the effect of nanosecond channel
dynamics on proton movement.
Short- and long-range dipolar interactions play an essential
role in biological ion transport. In gramicidin, solvation of
protonated water molecules and of bonding defects is achieved
locally by hydrogen bonds.6,7 In the single-file region of
aquaporin and water-glycerol channels,25-27 permeating water
molecules likewise form hydrogen bonds with backbone car-
bonyl O atoms, yet these channels are impermeable to H+.
Proton exclusion could be due in part to the adverse polarization
of the water chain induced by dipole-dipole interactions with
R helices lining the lumen.28 The dipole moment of four R
helices was shown to control charge selectivity in the potassium
channel KcsA.29 In the diffusion of K+ ions through the
selectivity filter of KcsA30 and of alkali metal ions through
gramicidin,8 backbone carbonyl groups directly solvate the
permeating ion via charge-dipole interactions. In the RR linked
gramicidin dimer, the dipole moment of the channel backbone
is modulated by thermal fluctuations. In the present work, we
show how these dynamic effects interfere with the flow of
protons, revealing the interplay between structural diffusion and
structural fluctuations of the proton conduit and underlining the
central importance of a fluctuating polar environment in charge
translocation. The biological significance of this result extends
to protein-mediated transport of protons in energy transduction.
This work demonstrates that the control of proton translocation
can be achieved by subtle modulations of the structure and
dynamics of the proteic matrix. Such effects could have
significant implications to molecular mechanisms of proton
pumping, where coupling between thermodynamic control of
proton movement by changes in the charge distribution in the
enzyme and kinetic control by structural rearrangement of proton
pathways31 is paramount.
Acknowledgment. This work was supported in parts by the
NIH (Grant RO1 GM59674) and by the Hospital for Sick
Children Research Institute. R.P. is a CRCP Chairholder.
JA0353208
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(24) Gowen, J. A.; Markham, J. C.; Morrison, S. E.; Cross, T. A.; Busath, D.
D.; Mapes, E. J.; Schumaker, M. F. Biophys. J. 2002, 83, 880.
(25) Fu, D. X.; Libson, A.; Miercke, L. J. W.; Weitzman, C.; Nollert, P.;
Krucinski, J.; Stroud, R. M. Science 2000, 290, 481.
(26) Sui, H.; Han, B. G.; Lee, J. K.; Walian, P.; Jap, B. K. Nature 2001, 414,
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A R T I C L E S Yu and Pome`s
13894 J. AM. CHEM. SOC. 9 VOL. 125, NO. 45, 2003