Atomic determinants of state-dependent block of sodium channels by charged local anesthetics and benzocaine.
ABSTRACT Molecular modeling predicts that a local anesthetic (LA) lidocaine binds to the resting and open Na(v)1.5 in different modes, interacting with LA-sensing residues known from experiments. Besides the major pathway via the open activation gate, LAs can reach the inner pore via a "sidewalk" between D3S6, D4S6, and D3P. The ammonium group of a cationic LA binds in the focus of the pore-helices macrodipoles, which also stabilize a Na(+) ion chelated by two benzocaine molecules. The LA's cationic group and a Na(+) ion in the selectivity filter repel each other suggesting that the Na(+) depletion upon slow inactivation would stabilize a LA, while a LA would stabilize slow-inactivated states.
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ABSTRACT: Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure-activity relationships, and effects of mutations in these ion channels.Trends in Pharmacological Sciences 01/2013; · 9.25 Impact Factor
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ABSTRACT: The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic "blocking particle". A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor
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ABSTRACT: The antianginal drug ranolazine, which combines inhibitory actions on rapid and sustained sodium currents with inhibition of the hERG/IKr potassium channel, shows promise as an antiarrhythmic agent. This study investigated the structural basis of hERG block by ranolazine, with lidocaine used as a low-potency, structurally similar comparator. Recordings of hERG current (IhERG) were made from cell lines expressing wild-type (WT) or mutant hERG channels. Docking simulations were performed using homology models built on MthK and KvAP templates. In conventional voltage clamp, ranolazine inhibited IhERG with an IC50 of 8.03μM; peak IhERG during ventricular action potential clamp was inhibited ~62% at 10μM. The IC50 values for ranolazine inhibition of the S620T inactivation-deficient and N588K attenuated-inactivation mutants were respectively ~73-fold and ~15-fold that for WT IhERG. Mutations near the bottom of the selectivity filter (V625A, S624A, T623A) exhibited IC50s between ~8 and 19-fold that for WT IhERG, whilst the Y652A and F656A S6 mutations had IC50s ~22-fold and 53-fold WT controls. Low potency lidocaine was comparatively insensitive to both pore-helix and S6 mutations, but was sensitive to direction of K(+) flux and particularly to loss of inactivation, with an IC50 for S620T-hERG ~49-fold that for WT IhERG. Docking simulations indicated that the larger size of ranolazine gives it potential for a greater range of interactions with hERG pore side chains compared to lidocaine, in particular enabling interaction of its two aromatic groups with side chains of both Y652 and F656. The N588K mutation is responsible for the SQT1 variant of short QT syndrome and our data suggest that ranolazine is unlikely to be effective against IKr/hERG in SQT1 patients.Journal of Molecular and Cellular Cardiology 05/2014; · 5.15 Impact Factor
Atomic determinants of state-dependent block of sodium channels
by charged local anesthetics and benzocaine
Denis B. Tikhonov, Iva Bruhova, Boris S. Zhorov*
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5
Received 4 September 2006; revised 11 October 2006; accepted 12 October 2006
Available online 24 October 2006
Edited by Maurice Montal
(LA) lidocaine binds to the resting and open Nav1.5 in different
modes, interacting with LA-sensing residues known from exper-
iments. Besides the major pathway via the open activation gate,
LAs can reach the inner pore via a ‘‘sidewalk’’ between D3S6,
D4S6, and D3P. The ammonium group of a cationic LA binds
in the focus of the pore-helices macrodipoles, which also stabilize
a Na+ion chelated by two benzocaine molecules. The LA’s cat-
ionic group and a Na+ion in the selectivity filter repel each other
suggesting that the Na+depletion upon slow inactivation would
stabilize a LA, while a LA would stabilize slow-inactivated
? 2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Molecular modeling predicts that a local anesthetic
Keywords: Homology modeling; Lidocaine; QX-314;
Monte Carlo-minimization; Ion permeation; Channel block;
Local anesthetics (LAs) are classical blockers of voltage-
gated sodium channels. LAs produce three main effects on
the channels: resting block, use-dependent block, and the shift
of inactivation [1,2]. To explain these effects, the ‘‘modulated-
receptor’’ hypothesis was elaborated . According to this
hypothesis, LAs slowly enter the closed channel through a
hydrophobic pathway. When the channel is activated by mem-
brane depolarization, the fast hydrophilic pathway becomes
open. The binding of LAs blocks ion permeation. LAs have
the highest affinity to the inactivated state and further stabilize
the inactivated state.
Mutational experiments revealed that LAs bind inside the
inner pore. LA-sensing residues were found in segments
D1S6, D3S6, and D4S6 [4,5]. It should be noted that effects
of mutations on the resting and use-dependent block are un-
Except for benzocaine and its analogs, LAs are protonated
or permanently charged molecules. Intriguingly, neutral benzo-
caine produces generally the same effects on the channel as
charged LAs. Benzocaine shares a common binding site with
other LAs, but some mutations have different influence on
the effects of benzocaine and etidocaine . Benzocaine does
not demonstrate a use-dependent block, but this peculiarity
is likely due to its fast kinetics . The fundamental difference
between benzocaine and other LAs is the Hill coefficient,
which in the case of benzocaine is not equal to 1 .
In this work, we use homology modeling and ligand docking
to address the following questions. What are energetically opti-
mal binding modes of LAs in the open and resting channels?
Whether a relatively large molecule like QX-314 could pass
to the inner pore of the resting channel between D3S6 and
D4S6, some residues in which had been shown to affect the
hydrophobic pathway? Why do so different molecules as
charged LAs and neutral benzocaine act similarly on sodium
channels? How slow inactivation could stabilize the binding
of both charged LAs and neutral benzocaine? Why do LAs sta-
bilize slow-inactivated states?
2. Model building and ligand docking
In the absence of X-ray structures of Na+channels, the
atomic determinants of the LA receptor can only emerge from
molecular models based on the X-ray structures of potassium
channels. We have built homology models of Nav1.5 (rH1) in
the open and closed states using, respectively, KvAP  and
KcsA  structures. The models were composed of P-loops
and S6 segments, whose sequences were aligned with K+chan-
nels as proposed earlier . For the selectivity-filter region,
which is significantly different between Na+and K+channels,
we used our earlier model of the P-loop domain, which was
shaped around tetrodotoxin and saxitoxin .
The models were optimized by the Monte Carlo-minimiza-
tion protocol as described elsewhere . To prevent large
deviations of the models from the templates, ‘‘pin’’ constraints
were imposed on Caatoms of a-helices. The optimal binding
modes of LAs were searched by a two-stage random-docking
approach. In the first stage, 30000 binding modes of a ligand
were randomly generated and MC-minimized in short trajecto-
ries of 20 energy minimizations. A thousand of the energeti-
cally best structures found at this stage were further
Abbreviations: LA, local anesthetic; MC, Monte Carlo; DEKA, the
circular locus of residues Asp, Glu, Lys, and Ala from P-loops in
domains D1–D4, respectively, which form the Na+channel selectivity
filter; D1S6–D4S6, the inner helices in domains D1–D4, respectively;
D3P, the pore helix in domain 3
*Corresponding author. Fax: +1 905 522 9033.
E-mail address: firstname.lastname@example.org (B.S. Zhorov).
0014-5793/$32.00 ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 580 (2006) 6027–6032
optimized in long MCM trajectories, each of which terminated
when the last 1000 energy minimizations did not improve the
apparent global minimum. Both ligand and protein were kept
flexible during energy minimizations. Henceforth, we use the
term ‘‘docking’’ to refer to the above procedure of searching
energetically optimal binding modes of the ligand. Thus, all
models discussed in this work represent stable low-energy con-
Although results of in silico experiments with homology
models are always questionable, the obtained models integrate
seemingly controversial experimental data on the action of
LAs in Na+channels and provide answers to the questions for-
mulated in the Introduction.
3. Ion dependence of LA binding
We have built four models of Nav1.5 in which the activation
gate was either open or closed, and the circular locus of res-
idues Asp, Glu, Lys, and Ala from P-loops in domains D1–
D4, respectively, which form the Na+channel selectivity filter
(DEKA) ring was loaded with either a Na+ion (Na+-DEKA)
or a water molecule (H2O-DEKA). A water molecule was
placed in the model to stabilize side-chain conformations of
the DEKA ring. Docking lidocaine, QX-314, and other
cationic LAs in the closed and open H2O-DEKA models pre-
dicted several binding modes in which the ammonium group
occurs at the focus of the pore-helix macrodipoles (Fig. 1A).
In this respect, Nav1.5-bound LAs resemble KcsA-bound
tetrabutylammonium, whose nitrogen is close to position 5
of K+in the ligand-free channel . In the open and closed
Na+-DEKA models, there is a significant electrostatic repul-
sion between the ammonium group of the ligand and the
Na+ion. The repulsion weakens the LA-channel interaction
energy and moves the ligand’s ammonium group away from
the focus of helical macrodipoles (Fig. 1B). These results sug-
gest that LAs should have higher affinity to channels that lack
Na+in the DEKA locus and that the binding of LAs should
prevent Na+to occupy the DEKA locus. Based on this result,
we performed other computational experiments on the H2O-
The docking yielded two significantly different binding
modes of LAs: planar and axial (Fig. 2). In the planar mode,
the aromatic ring of the ligand protrudes between D3S6 and
D4S6. In the axial mode, the aromatic ring faces intracellu-
larly. Importantly, in both modes, F1762 and Y1769 in
D4S6 (F1764 and Y1771 in rat brain IIA channel; F1579
and Y1586 in rat skeletal muscle isoform), which were identi-
fied in mutational studies as critical determinants of the LA
receptor, are significant contributors to the ligand binding en-
ergy. Segments D1S6 and D3S6 provide modest contributions,
whereas the interactions of LAs with D2S6 are very weak. In
both binding modes, the ammonium group of the ligand is
located at the focus of helical macrodipoles. The axial binding
mode coincides with the proposal based on mutational and
ligand-binding studies  and with a recent model of use-
dependent block . A possibility of an alternative planar
binding mode, impact of helical dipoles, and the role of ions
in the selectivity filter are new proposals.
4. LA access and binding in the resting and open channels
In the open channel, the obvious access of LAs to the inner
pore is through the widely open activation gates. In the closed
state, this pathway is impermeable for LAs. Some residues in
S6s are known to affect the resting-channel block by the exter-
nal quaternary LAs [17,18]. Mapping these residues in the
model Nav1.4, visualizes a ‘‘sidewalk’’ to the inner pore be-
tween D3S6 and D4S6 along the pore helix in domain 3
(D3P) . A Cys mutant of Nav1.5, whose position is imme-
diately C-terminal to Asp of the DEKA locus, affects egress of
the protonated lidocaine from the pore suggesting that a
hydrophilic pathway via the outer pore may coexist with the
Fig. 1. The side views of the lowest-energy complexes of lidocaine in H2O-DEKA (A) and Na+-DEKA (B) models of the open Nav1.5. The inner
helices and pore helices are shown as violet and green ribbons, respectively. Domain 2 is removed for clarity. The P-loop segments lining the outer
pore are shown by Catracings. Lidocaine is shown by thick sticks. Side chains of the DEKA locus are shown by thin sticks. Side chains of Phe and
Tyr residues in D4S6, which are critical determinants of the LA receptor, are colored magenta. The water molecule and Na+ion at the DEKA locus
are shown by red and yellow spheres, respectively. In the H2O-DEKA model (A), which represents a slow-inactivated channel, the ammonium group
of the ligand is stabilized at the focus of macrodipoles of the pore helices. In the Na+-DEKA model (B), the repulsion between the Na+ion and the
ligand’s ammonium group cause the latter to face the cytoplasmic entrance to the open pore.
D.B. Tikhonov et al. / FEBS Letters 580 (2006) 6027–6032
hydrophobic pathway . In this work, we explored only the
hydrophobic pathway, which is lined by the protein segments
whose mutual disposition is defined in the X-ray structures
of the K+channel templates.
We have pulled QX-314 along the hydrophobic pathway
with the step of 1 A˚and MC-minimizing the energy at each
step. The superposition of the MC-minimized structures with
the ligand at different positions along the hydrophobic path-
way is shown in Fig. 3. No high-energy barriers were predicted
in these computations suggesting that the hydrophobic path-
way is passable by QX-314 and hence by smaller LAs. Exact
molecular mechanisms of isoform specificity of QX-314 access
and effects of mutations need systematic study. A possible dif-
ference in the access of the protonated and deprotonated forms
of LAs also should be addressed in future works. For example,
since the hydrophobic pathway is close to the selectivity filter,
presence of an ion in the DEKA locus could affect access of
Our results suggest the following mechanism of LA action.
A deprotonated LA molecule dissolved in the membrane
would reach the resting-channel inner pore through the path-
way between D3S6, D4S6, and D3P. Inside the pore, the LA
molecule would accept a proton and become a hydrophobic
cation enjoying hydrophobic interactions with hydrophobic
residues in the inner helices and electrostatic interactions with
the pore-helices macrodipoles. In the cardiac channel, quater-
nary compounds also can enter the channel via this pathway.
Importantly, the lowest-energy structures obtained at the
inner-pore end of the hydrophobic pathway (Fig. 3) are very
similar to the planar binding mode of LAs found by random
docking (Fig. 2). Since the closed inner pore does not provide
enough room for a LA molecule to maneuver, the molecule
that enters the pore via the hydrophobic pathway would re-
main in the planar binding mode. In the open channel, LAs
reach the binding site rapidly via the open intracellular gate
and, most likely, would bind in the axial mode, which is
Fig. 2. The cytoplasmic (A,C) and side (B,D) views of lidocaine in the H2O-DEKA models of the open (A,B) and closed (C,D) Nav1.5. In both the
open and closed channels, the ligand interacts with Phe and Tyr residues of D4S6 (magenta sticks). In the open channel, the ligand is in the axial
binding mode, which is complementary to the open inner pore. In the closed channel, the ligand is in the planar binding mode. In both open and
closed channels, the ligand’s ammonium group binds at the focus of the macrodipoles of the pore helices. For clarity the side views have a transparent
D.B. Tikhonov et al. / FEBS Letters 580 (2006) 6027–6032
complementary to the open pore. Thus, the binding mode of
LAs in the open and closed channels may depend on the access
pathway. This model explains the observation that mutations
of F1762 and Y1769 in D4S6 cause qualitatively different effect
on the use-dependent and resting block .
5. Relations between slow inactivation and binding of cationic
Our models provide an explanation to the notion that cat-
ionic LAs bind preferably to slow-inactivated channels .
The presence of a Na+ion in the DEKA locus would destabi-
lize the binding of a cationic ligand. Slow-inactivated channels
do not permeate ions and therefore may lack Na+in the
DEKA locus. X-ray structures suggest that the extracellular
gate closure in K+channels may be associated with the ion
deficiency in the selectivity filter [14,20]. The binding of cat-
ionic ligands in the inner pore of K+channels enhances slow
inactivation [21,22]. The cause of this phenomenon may be
the depletion of a K+ion from the selectivity filter by a cationic
ligand . Berneche and Roux  suggested that the rear-
rangements of the selectivity filter in KcsA caused by ion defi-
ciency might be associated with slow inactivation.
By analogy with K+channels, we propose that the H2O-
DEKA and Na+-DEKA models correspond, respectively, to
the slow inactivated and conducting states of the Na+channel.
Docking LAs in the H2O-DEKA and Na+-DEKA models ex-
plains the state-dependent action of cationic ligands by repul-
sion between the ligand’s ammonium group and an ion in the
DEKA locus. Cationic LAs bind stronger to the slow-inacti-
vated (H2O-DEKA) channels and stabilize the slow-inacti-
vated state by antagonizing occupation of the DEKA ring
by an ion. Certainly, slow inactivation is a complex process
that probably involves yet unknown structural rearrangements
of the channel protein. Our simple model of the slow-inacti-
vated state just suggests that the DEKA locus occupancy by
either a cation or a water molecule may be of critical impor-
tance for the state-dependent ligand binding.
6. The benzocaine paradox
Cationic ligands block the ion permeation by occupying
the focus of macrodipoles of the pore helices. From this point
of view, neutral benzocaine seems like a puzzle because the
binding of a small amphiphilic molecule is not expected to
block ion permeation. The shift of steady-state inactivation
by benzocaine  is also a puzzle. Our models explain the
channel block and effect of inactivation by the antagonism be-
tween Na+in the DEKA locus and the ammonium group in
the focus of the macrodipoles, but benzocaine lacks a charged
A key to resolve the paradox is the assumption that the ester
group of benzocaine binds a Na+ion. Earlier we proposed a
model, in which the sodium channel agonist batrachotoxin
occupies the inner pore close to the selectivity filter and a per-
meant Na+ion directly binds to oxygen atoms of the ligand
. A touchstone prediction of the model was that Phe pre-
ceding Lys of the DEKA locus contributes to the batracho-
toxin receptor. Recent experiments confirmed the prediction
. Since the Hill coefficient for benzocaine binding is greater
than 1, we further suggest that two benzocaine molecules inter-
act with a Na+ion inside the pore. To explore this possibility
computationally, we generated a large number of starting
points with two benzocaine molecules and a Na+ion randomly
seeded in a large sphere volume in the inner pore of the closed
channel. The two-stage random docking protocol (see 2)
yielded lowest-energy structures with two benzocaine mole-
cules jointly chelating the Na+ion by their ester groups. One
of such structures (Fig. 4) shows that the Na+ion bound to
benzocaine can mimic the ammonium group of a charged
LA. One benzocaine molecule occurs in the planar, and
another in the axial orientation in the channel, while aro-
Fig. 3. Moving QX-314 to the closed Nav1.5 via the hydrophobic pathway between D3S6, D4S6, and D3P. Panels A and B show orthogonal side
views at the superposition of MC-minimized structures with the ligand constrained at different distances from the pore axis. Residues in the
extracellular half of D4S6, which affect the Na+channel block by the extracellularly applied LAs with the quaternary ammonium group [17,18], are
space-filled and magenta-colored.
D.B. Tikhonov et al. / FEBS Letters 580 (2006) 6027–6032
matic rings interact with critical F1762 and Y1769 residues in
The concentration dependence of benzocaine block is com-
plex: the Hill coefficient increases with the benzocaine concen-
dependence was not explained. In the view of our model, at
low concentrations one molecule of benzocaine can bind to
the Na+ion in the focus of macrodipoles, stabilize it, and
block ion permeation. At high concentrations, two benzocaine
molecules would jointly chelate the Na+ion.
Benzocaine binding is not a unique example of a concentra-
tion-dependent Hill coefficient. Similar concentration depen-
dence was observed with S-nitrosodithiothreitol action on
Kv1 channels . The authors of the study suggested that
two S-nitrosodithiothreitol molecules bind to the channel,
but even a single molecule can block it. A notable analogy with
benzocaine is that S-nitrosodithiothreitol is a small, uncharged
molecule, which has nothing in common with a typical cationic
blocker of K+channels, tetraethylammonium. Like benzo-
caine, S-nitrosodithiothreitol has polar groups that could bind
to a permeant cation inside the pore and stabilize it. Indeed,
the voltage dependence of S-nitrosodithiothreitol action
suggests that K+forms a charged complex with the ligand(s),
and that this complex blocks the channel.
7. Concluding remarks
Results of molecular modeling of the cardiac Na+channel
and its complexes with LAs provide new insights to the prob-
lem of state-dependent ligand action. Our study integrates var-
ious experimental data and suggests a hypothesis on the
common mechanism of the channel block by essentially differ-
ent ligands and on molecular interactions underlying relations
between the slow inactivation and the channel block. Consid-
erable efforts are needed to experimentally verify the hypothe-
sis, which integrates data from decades of experimental
studies. If correct, the hypothesis could be of interest beyond
the sodium channels field. Further analysis of existent and
new experimental data in view of our models is necessary.
Coordinates of the models are available upon request from
Acknowledgements: This work was supported by the grant from
the Canadian Institutes of Health Research. Computations were
performed, in part, using the facilities of the Shared Hierarchical
Academic Research Computing Network (SHARCNET: www.
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D.B. Tikhonov et al. / FEBS Letters 580 (2006) 6027–6032