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.
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