Supplemental Material can be found at:

http://www.jbc.org/content/suppl/2007/08/08/M704651200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 40, pp. 29424 29430, October 5, 2007
2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Design of a Specific Activator for Skeletal Muscle Sodium

Channels Uncovers Channel Architecture*
S

Received for publication, June 6, 2007, and in revised form, August 6, 2007 Published, JBC Papers in Press, August 8, 2007, DOI 10.1074/jbc.M704651200

Lior Cohen, Nitza Ilan, Maya Gur, Walter Stuhmer, Dalia Gordon1, and Michael Gurevitz2
From the Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv,
69978 Tel-Aviv, Israel and Department of Molecular Biology and Neuronal Signaling, Max Planck Institute of Experimental
Medicine, D-37075 Gottingen, Germany

* This

work was supported by German-Israeli Foundation for Scientific

Research and Development Grant G-770-242.1/2002 (to D. G. and W. S.), by
United States-Israel Binational Agricultural Research and Development
Grant IS-3480-03 (to M. G. and D. G.), by Israeli Science Foundation Grants
733/01 (to M. G.) and 1008/05 (to D. G.), and by National Institutes of Health
Grant 1 U01 NS058039-01 (to M. G.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.

S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1 and S2 and Table S1.
1
To whom correspondence may be addressed. Tel.: 972-3-6409844; Fax: 9723-6406100; E-mail: dgordon@post.tau.ac.il.
2
To whom correspondence may be addressed. E-mail: mamgur@post.
tau.ac.il.

29424 JOURNAL OF BIOLOGICAL CHEMISTRY

Among the superfamily of voltage-gated ion channels,

sodium channels (Navs)3 play a central role in the generation of
action potentials in excitable cells and are the target of a large
variety of neurotoxins, drugs, and insecticides (1, 2). Although
the structure of homo-tetrameric voltage-gated potassium
channels (Kvs) was recently resolved (3, 4), the structure of Navs
has not yet been determined.
Eukaryotic Navs are constituted from one large protein that
forms four homologous, non-identical domains. As for Kvs,
each domain of the Nav comprises two functional modules. The
pore module consists of transmembrane segments S5 and S6
connected by a membrane-associated re-entrant loop (SS1SS2). The voltage-sensing module, which consists of segments
S1-S4, undergoes a conformational alteration in response to
changes in the membrane potential, enabling channel activation (1, 5). Estimation of the distances between transmembrane
segments of the homo-tetrameric bacterial Nav, NaChBac, suggested an intra-subunit organization similar to that of Kvs in a
lipid bilayer (6), but the inter-domain organization of the pore
and voltage-sensing modules in eukaryotic Navs have not been
determined.
A variety of mammalian Navs, encoded by at least nine
genes, have been described (1, 7). Expression of these channels varies greatly across tissues and developmental stages
(1). Nav1.11.3 and Nav1.6 are expressed in the central nervous system, Nav1.6 and Nav1.7 are expressed in the peripheral nervous system, Nav1.8 and Nav1.9 are expressed in sensory neurons, and Nav1.4 and Nav1.5 are expressed in
skeletal and cardiac muscle, respectively. Many genetic disorders leading to abnormal function of these channels have
been described in humans (e.g. Refs. 8 12). For example,
mutations in the SCN4A gene, encoding for the human skeletal muscle channel Nav1.4, lead to various types of periodic
paralysis, paramyotonia congenital, and Nav myotonias
(reviewed in Ref. 8). Several mutations rendering hypokalemic periodic paralysis (hypoPP) (8, 1315) were identified in
the voltage sensor segment S4 of Domain 2 (D2/S4) of the
human skeletal muscle sodium channel, hNav1.4, causing a
positive shift in the voltage dependence of channel activation
and gating pore currents at resting membrane potential (13
16). Action potentials evoked from human hypoPP muscle
samples are sluggish and smaller than those obtained in
3

The abbreviations used are: Nav, voltage-gated sodium channel; Css4, Centruroides suffusus suffusus -toxin 4; hypoPP, hypokalemic periodic
paralysis.

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Gating modifiers of voltage-gated sodium channels (Navs)

are important tools in neuroscience research and may have
therapeutic potential in medicinal disorders. Analysis of the
bioactive surface of the scorpion -toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNav1.2a) and skeletal muscle (rNav1.4) channels using binding studies revealed
commonality but also substantial differences, which were
used to design a specific activator, Css4F14A/E15A/E28R, of
rNav1.4 expressed in Xenopus oocytes. The therapeutic
potential of Css4F14A/E15A/E28R was tested using an rNav1.4
mutant carrying the same mutation present in the genetic
disorder hypokalemic periodic paralysis. The activator
restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means
for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin
residues involved in the construction of Css4F14A/E15A/E28R
and residues whose equivalents in the rat brain channel
rNav1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and
E592A at Domain-2/voltage sensor segments 12 (D2/S1-S2),
R27Q and E1251N at D3/SS2-S6, and E28R with both E650A
at D2/S3-S4 and E1251N at D3/SS2-S6. These results show
that despite the differences in interactions with the rat brain
and skeletal muscle Navs, Css4 recognizes a similar region on
both channel subtypes. Moreover, our data indicate that the
S3-S4 loop of the voltage sensor module in Domain-2 is in very
close proximity to the SS2-S6 segment of the pore module of
Domain-3 in rNav1.4. This is the first experimental evidence
that the inter-domain spatial organization of mammalian Navs
resembles that of voltage-gated potassium channels.

Nav1.4 Architecture Uncovered by a Specific Activator

OCTOBER 5, 2007 VOLUME 282 NUMBER 40

of the gating and pore modules of two adjacent Nav domains

and provide evidence that the inter-domain architecture of
Navs resembles that of Kvs.

EXPERIMENTAL PROCEDURES
Mutagenesis of Css4PCR-driven mutagenesis, expression
in Escherichia coli, in vitro folding, and purification of Css4
derivatives have been described in detail elsewhere (25).
Expression of Sodium Channels in OocytesThe pNa200
vectors bearing the genes encoding rNav1.2a, rNav1.3, and
rNav1.6 were a gift from Dr. A. Goldin (University of California,
Irvine). The pAlter vectors bearing the genes encoding rNav1.4
and hNav1.5 were a gift from Dr. R. G. Kallen (University of
Pennsylvania, Philadelphia, PA). These genes and the auxiliary
h1 were transcribed in vitro using T7 RNA polymerase and
the mMESSAGE mMACHINETM system (Ambion, Austin,
TX) (27, 28) and injected into Xenopus laevis oocytes as previously described (21).
Site-directed Mutagenesis of rNav1.4pAlter containing the
entire rat skeletal muscle sodium channel -subunit was used
for oligonucleotide-based mutagenesis. The PCR-amplified
fragment containing the mutations E592A, H599Q/D601S/
N602S, E650A, L653A, Q657E, G658N, and R666G was
cleaved by BsiwI and BssHII and inserted into the corresponding sites of the vector. E1251N and H1257K were
cleaved by EcoNI and inserted into the corresponding sites
of the vector, which was further used for transcription after
DNA sequence verification.
Two Electrode Voltage Clamp Recording and Data Analysis
Currents were measured 12 days after injection using a two
electrode voltage clamp and a Gene Clamp 500 amplifier
(Axon Instruments, Union City, CA). Data were sampled at
10 kHz and filtered at 5 kHz. Toxins were diluted with bath
solution and applied directly to the bath to achieve the
desired final concentration. Data acquisition was controlled
by a Macintosh PPC 7100/80 computer equipped with an
ITC-16 analog/digital converter (Instrutech Corp., Port
Washington, NY), using Synapse (Synergistic Systems). The
bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, and 5 mM HEPES, pH 7.85. Oocytes were
washed with bath solution flowing from a BPS-8 perfusion
system (ALA Scientific Instruments, Westbury, NY) with a
positive pressure of 4 psi.
Capacitance transients and leak currents were removed by
subtracting a scaled control trace using a P/6 protocol (29).
For the GV analysis, mean conductance (G) was calculated
from the peak current/voltage relationship using the equation G I/(V Vrev), where I is the peak current, V is the
membrane potential, and Vrev is the reversal potential. The
normalized conductance/voltage relationship was fit with
either a one- or two-component Boltzmann distribution
according to Equation 1
G/Gmax (1 A)/(1 exp[(V112 V)/k1]) A/(1
exp[(V212 V)/k2])
where V112 and V212 are the respective membrane potentials
for two populations of channels for which the mean conductance is half maximal, k1 and k2 are their respective slopes, and A
defines the proportion of the second population (amplitude)
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healthy muscle tissue (15). Gating modifiers that facilitate

channel activation could potentially restore the properties of
defective Navs, but to be therapeutically applicable they have
to be highly specific for hNav1.4.
Scorpion -toxins (e.g. the anti-mammalian Css2 and Css4
from Centruroides suffusus suffusus) are short polypeptides
reticulated by four disulfide bonds that target Navs and modulate their gating properties (17). They are typified by the
shift they induce in the voltage dependence of channel activation to more negative membrane potentials upon binding
to receptor site 4 (17), shown to be associated with Domain 2
of the Nav (18 21). Four conserved residues in D2/S1-S2 and
S3-S4 have been implicated in the interaction of the -toxin
Css4 with mammalian Navs (19, 20), and two residues in
D3/SS2-S6 have recently been linked to the preference of the
-toxin Tz1, from the scorpion Tityus zulianus, for the muscle channel rNav1.4 over the brain channel rNav1.2a (22).
These data, and the fact that Css4 is more potent at hNav1.2a
than hNav1.4 (23), have suggested that the set of residues
comprising receptor site 4 on both channels are not
identical.
As the binding of -toxins is independent of membrane
potential and because a depolarizing prepulse is required to
observe their effect, it was suggested that prebound -toxins
trap the D2/S4 segment in its outward activated position,
leading to enhanced channel activation upon subsequent
depolarizations (19). Despite the accumulated knowledge
about their mode of interaction with the Nav, complete elucidation of neurotoxin receptor site 4 and the extent of its
conservation in various channel subtypes remained to be
described.
Toxins with high preference for Nav subtypes are useful for
identifying distinct channels and studying their mechanism of
action and may also be instrumental in development of new
drugs for treating neurological and muscular disorders. Moreover, as the receptor binding sites for toxins incorporate residues of different, not necessarily adjacent, extracellular loops
(1, 20, 22), by identifying interacting residues of toxins and
channels and by relying on the known toxin structures we may
elucidate the spatial organization of the channel extracellular
face.
Mutagenic dissection of scorpion -toxins that affect both
mammalian (Css4) and insect (the excitatory toxin Bj-xtrIT
from Buthotus judaicus and the depressant toxin LqhIT2 from
Leiurus quinquestriatus hebraeus) Navs revealed a spatially
similar cluster of bioactive residues (24 26). Moreover, we
have suggested that a spatially conserved Glu residue in -toxins (position 28 in Css4) forms a hot spot for interaction with
receptor site 4 (25).
Here we show profound variations in the bioactive surface of
Css4 toward the rat muscle and brain Navs, which we used to
construct a Css4 triple mutant that exclusively binds and affects
rNav1.4. This mutant toxin can restore most of the gating properties of a rNav1.4 mutant designed according to a mutation
found in hypoPP patients. We also identified Css4-rNav1.4 residue pairs that exhibit coupling energies indicating close proximity of specific extracellular channel loops. These data enabled
the first estimation of the distance between extracellular loops

Nav1.4 Architecture Uncovered by a Specific Activator

RESULTS
Comparison of Css4 Interactions with Rat Skeletal Muscle
and Brain Navs and Construction of a Specific Modifier for
Nav1.4To clarify the difference in Css4 potency at Nav1.2a
versus Nav1.4 we sought for the toxin bioactive surface
toward the skeletal muscle Nav and compared it with the
reported surface toward brain Navs (25). We have previously
shown that substitution of Tyr-4, Thr-10, Phe-14, Leu-19,
Tyr-24, Arg-27, Glu-28, Gln-32, Tyr-40, Tyr-42, Phe-44,
Trp-47, Trp-58, and Lys-63 for Ala considerably affected
Css4 binding affinity for rat brain synaptosomes (supplemental Table S1 and Ref. 25). Most of these substitutions
also decreased the binding affinity for rat muscle membranes, except for those at Thr-10, Phe-14, Glu-28, Gln-32,
and Lys-63 (supplemental Table S1 and Fig. 1). These results
raised a subset of residues that are involved in the surface of
interaction of the toxin with both rat brain and skeletal muscle Navs and highlighted a few residues whose roles differ.
This difference is notable in light of the CD alteration ren-

29426 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 1. Competition of wild-type and mutant Css4 toxins on binding to

rat brain and skeletal muscle membrane preparations. Membranes were
incubated with 0.1 nM 125I-His-Css4 and increasing concentrations of the indicated mutants at 22 C for 60 min. Nonspecific binding, determined in the
presence of 1 M His-Css4, was subtracted. The Ki values (in nM, n 3) for rat
brain synaptosomes and rat skeletal muscle membranes, respectively, were
Css4, 0.98 0.1, 3.9 1.17; Css4E15A, 0.07 0.01, 0.3 0.1; Css4F14A, 141 18,
3.7 0.4; Css4R27A, 31 6.3, 56.5 10.5; Css4E28A, 635 98, 6.2 2.3. The
curves are from representative experiments.

dered by T10A, F14A, and K63A and the fact that Glu-28 and
Gln-32 have been assigned to the hot spot in the surface of
interaction of Css4 with the rat brain channel (25). Thus,
receptor site-4, which pertains to the interface between Css4
and the Nav, differs in mammalian brain versus muscle Navs,
especially in respect to Phe-14 and Glu-28 (supplemental
Table S1), which laid the groundwork for construction of a
mutant toxin specific for Nav1.4. Because the affinity of
mutant E15A for both channel types exceeded 10-fold that of
the unmodified toxin (supplemental Table 1 and Fig. 1), we
constructed Css4 mutants bearing various combinations of
substitutions F14A and E28R in the background of E15A.
The most significant among these constructs was the triple
mutant Css4F14A/E15A/E28R, whose affinity for the brain channel dropped 254-fold whereas its affinity for the skeletal
muscle channel was similar to that of Css4E15A (supplemental Table S1), thus providing a highly selective ligand for
muscle channels.
We then analyzed the effects of these mutants on a variety of
mammalian Navs co-expressed in Xenopus oocytes with the 1
subunit, using Css4E15A as the first point of reference. Css4E15A
was active at rNav1.2a and rNav1.4 but had no effect at rNav1.3,
hNav1.5, and rNav1.6 even at a concentration of 5 M (Table 1
and supplemental Fig. S1). Hence, the preference of Css4E15A
for the mammalian Nav subtypes was similar to that of Css4 (19,
23, 33), yet the potency of Css4E15A increased 10-fold (EC50
0.46 for rNav1.2a and 0.88 M for rNav1.4) compared with the
unmodified toxin (25). Css4E15A induced a 25-mV shift in the
threshold of activation for both rNav1.2a and rNav1.4 (at 0.5
and 1 M, respectively) (Fig. 2 and supplemental Fig. S1). The
conductance-voltage relation (G/V) curves for rNav1.2a and
rNav1.4 in the presence of Css4E15A exhibit two components
with a minor negative shift in the voltage for half activation
(V0.5) of the entire channel population and a stronger shift in
the V0.5 of a fraction of the channel population (Fig. 2 and Table
1, values in parentheses). The effects of Css4E15A on channel
activation resembled those of Css4 in the requirement for a
depolarizing prepulse of similar duration and the rates of assoVOLUME 282 NUMBER 40 OCTOBER 5, 2007

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with respect to the total. For fits in which only one population of
channels was apparent, A was set to zero. The voltage dependence of steady-state fast inactivation was described using a single Boltzmann distribution as shown in Equation 2
I/Imax 0 1/(1 exp[(V V12)/k])
where I is the peak current measured during the test depolarization step, Imax is the current without a preceding conditioning step, V is the membrane potential of the conditioning
step, V12 is the membrane potential at which half-maximal inactivation is achieved, k is the slope factor, 0 is the remaining
normalized peak current at highly depolarizing conditioning
potentials, and 1 is the normalized amplitude (30). For mutant
double cycle analysis, the dose response for Css4 effects on
rNav1.4 was determined based on the current induced by the
toxin following a test pulse to a voltage of 35 mV from the V0.5
of activation determined for each channel mutant (Table 1).
The free energy change in toxin binding to the wild-type/mutant channel pair (G) was calculated as the difference of the
average RTln (EC50) for the wild type and mutant, where R is
the gas constant and T stands for temperature in 0K. G was
taken as the difference of the Gs for Css4 and the toxin
mutant: G (GWT, native Gmutant, native) (GWT, mutant
Gmutant, mutant), where the first and second subscript positions refer to the channel and the toxin, respectively. RT
0.59 kcal/mol.
Binding ExperimentsSkeletal muscle membranes and brain
synaptosomes were prepared from adult albino Wistar rats
(300 g, laboratory-bred) as previously described (31, 32). The
rats were sacrificed according to the rules of the Animal Care
Unit at Tel Aviv University (permit number L-03-54) following
National Institutes of Health guidelines. Radio-iodination of
Css4, purification of monoiodotoxin, and binding assays were
performed as previously described (25). Nonspecific toxin
binding was determined in the presence of 1 M unlabeled toxin
and consisted typically of 1530% of total binding. Each experiment was performed in duplicate and repeated at least three
times as indicated (n). Data are presented as the mean S.D.
for independent experiments.

Nav1.4 Architecture Uncovered by a Specific Activator

TABLE 1
Effects of Css4 mutants on the activation of various Navs and rNav1.4 mutants
The G/V relations in the presence of Css4 mutants exhibit two components: a minor negative shift in the V0.5 of activation of the entire channel population (upper
number), and a stronger shift in the V0.5 (lower number) of a fraction (in parentheses) of the channel population. Data represent the mean S.E. of at least six
independent experiments. The EC50 of Css4E15A was 0.46 and 0.88 M for rNav1.2a and rNav1.4, respectively. Current-voltage relations were determined as described
in Fig. 2. ND, not determined.
Css4
Channel

Control
V0.5 mV

E15A

V0.5 mV

rNav1.2a

24.9 0.4 0.5 29.6 0.5

42 3 (39%)

E15A/F14A

E15A/R27Q

E15A/E28R

V0.5 mV

V0.5 mV

10

25.3 0.3

10

25.1 0.2

10

26.1 0.3

V0.5 mV

F14A/E15A/E28R

M
10

V0.5 mV
25.2 1.2

rNav1.3

19.1 0.5

18.1 0.6

ND

ND

10

19.4 0.7

10

18.8 1.4

hNav1.5

35.1 0.3

35 0.5

ND

ND

10

35 0.4

10

35.1 0.4

rNav1.6

27.7 0.5

25.8 1.6

ND

10

26.3 0.4

10

26.7 0.9

rNav1.4

24.9 0.3

31 0.2
36.1 2.1 (21%)

31.4 0.4
40.1 2.5 (22%)

25

29.2 0.4
34.5 1.5 (12%)

31 0.2
36.3 1.6 (21%)

E592A

15.1 0.6

16.3 0.2
31 2.7 (25%)

20

16.2 0.4
30 2.1 (25%)

10

14.9 0.4

16.7 0.3
35.7 2.6 (28%)

10

15.1 0.3

H599Q/D601S/
N602S

25 0.2

25.3 0.2

25.3 0.2

10

24.4 0.7

24.3 1.2

E650A

20 0.4

22.5 0.5
43.2 1.7 (28%)

10

-20 0.3

30

21.3 0.3
37.7 2.1 (16%)

10

20.2 0.6

L653A

22.3 0.2

10

23.6 0.1

10

23.3 0.7

10

23.6 0.1

10

23.8 0.4

Q657E

25 0.2

26.1 0.3
41 3.1 (19%)

10

25.2 0.8

G658N

26.8 0.2

10

26.8 0.3

10

26.6 0.5

10

26.7 0.3

10

26.8 0.3

E1251N

27.2 0.5

29.5 0.5
52 2.3 (27%)

30 0.7
56 5.2 (26%)

29.7 0.5
52.3 3.1 (30%)

25

30.2 0.6
54 3.2 (25%)

10

26.8 0.8

H1257K

29.7 0.4

31.7 0.9
39.3 2.7 (17%)

31.5 0.2
38.7 2.1 (16%)

10

32.7 0.3
38.5 2.8 (14%)

10

31.7 0.5
39.1 1.8 (16%)

ND

33.8 3.9 (19%)

22.3 0.3
42.7 2.1 (30%)
23.6 0.1

26.8 0.3

10
1
10

FIGURE 2. Conductance-voltage relations for rNav1.2a and rNav1.4 in the

presence of various Css4 mutants. A, rNav1.4. B, rNav1.2a. Concentrations of
the mutant toxins and activation parameters (V0.5) are shown in Table 1. The
current-voltage relations were determined as described in supplemental Fig.
S1 using the voltage protocol with a depolarizing prepulse.

ciation and dissociation at rNav1.2a and rNav1.4 (supplemental

Fig. S1) (19, 20, 23).
Css4F14A/E15A, Css4E15A/E28R, and Css4F14A/E15A/E28R had no
effect on the voltage dependence of activation of the neuronal
channels rNav1.2a, rNav1.3, and rNav1.6 and the cardiac channel hNav1.5. However, they shifted the voltage dependence of
rNav1.4 activation equally as well as Css4E15A, demonstrating
complete specificity for the skeletal muscle channel (Table 1
and Fig. 2).
Effect of Css4F14A/E15A/E28R on the Gating Properties of
rNav1.4R666G, an Equivalent of the Genetic Disorder
hNav1.4R672GAn R672G mutation in D2/S4 of the human
Nav1.4 was identified in the SCN4A gene of patients with
hypoPP (8) and shown to generate an 8-mV rightward shift in the
OCTOBER 5, 2007 VOLUME 282 NUMBER 40

34.6 1.7 (21%)

31.1 0.2

26.4 0.9
46 1.6 (20%)

33.2 2.4 (21%)

26.3 1.3
43 2.1 (18%)

FIGURE 3. Characterization of rNav1.4R666G and Css4F14A/E15A/E28R effects

on channel gating properties. A, conductance-voltage relations of the
unmodified rNav1.4 (V0.5 24.9 0.3 mV) and of rNav1.4R666G in the
absence (V0.5 17 1.3 mV) and presence of 1 M Css4F14A/E15A/E28R (V0.5
25 0.7 and 34.2 1.3 mV for 13% of the channel population). The currentvoltage relations were determined as described in Fig. 2. B, steady-state inactivation of rNav1.4 fits a Boltzmann function (see Experimental Procedures, and
Equation 2) with V0.5 49 0.6 and V0.5 55.5 0.7 mV for rNav1.4R666G
and V0.5 50.4 0.7 mV for rNav1.4R666G in the presence of 1 M
Css4F14A/E15A/E28R. Steady-state fast inactivation was determined using a
50-ms prepulse to 60 mV followed by a hyperpolarizing pulse to 100 mV
and a series of 50-ms prepulses from 90 to 20 mV in 5-mV increments
before the test pulse of 20 mV (see inset).

voltage dependence of activation and a 5-mV leftward shift in the

steady-state fast inactivation (13). Because Css4F14A/E15A/E28R
induced a hyperpolarizing shift in the voltage dependence of
rNav1.4 activation (Fig. 2), we examined its effects on an identical mutation constructed in the equivalent position of the rat
Nav1.4 (rNav1.4R666G). We found that the voltage dependence
of channel activation indeed was right-shifted by 8 mV (V0.5
17 1.3 mV) relative to the unmodified channel (V0.5
24.9 0.3 mV, Fig. 3A), as well as its steady-state fast inactivation that was left-shifted by 5 mV (V0.5 55.5 0.7 mV)
relative to the unmodified channel (V0.5 49 0.6 mV, Fig.
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26.4 0.1
45 2 (18%)

34.2 2.2 (20%)

1

25 0.3

31.6 0.3
39.5 2.5 (24%)

Nav1.4 Architecture Uncovered by a Specific Activator

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was right-shifted, providing a V0.5

50.4 0.7 mV (Fig. 3B). These
effects by Css4F14A/E15A/E28R demonstrated its ability to restore most of
the altered gating properties of
rNav1.4R666G, which under the
influence of this specific modulator performed much like the
unmodified rNav1.4 under control
conditions.
Aside from the therapeutic
potential arising from the specificity
of Css4F14A/E15A/E28R for rNav1.4,
our results have raised the question
of whether Css4 recognizes a similar
region in rNav1.2a and rNav1.4.
Therefore, we examined the effects
of mutations in both the toxin and
the channel on their interaction.
Css4E15A Effects on rNav1.4
MutantsBased on recent reports
about substitutions introduced to
rNav1.2a (E779Q in D2/S1-S2;
E837Q, L840C, and G845N in
D2/S3-S4) and rNav1.4 (G658N in
D2/S3-S4; E1251N and H1257K
in D3/SS2-S6) that reduced the
effects of the -toxins Css4 (19, 20)
and Tz1 (22), we constructed
rNav1.4 mutants E592A in D2/S1S2; E650A, L653A, and G658N in
D2/S3-S4; and E1251N and H1257K
in D3/SS2-S6 (see Fig. 4A for
sequence alignment). In addition,
four residues that differ between
rNav1.2a and rNav1.4 at D2/S1-S2
FIGURE 4. Effects of Css4E15A on rNav1.4 mutants. A, alignment of D2/S1-S2, D2/S3-S4, and D3/SS2-S6 Nav regions and S3-S4 were substituted at
of a number of Navs (Swissprot accession numbers are P15390 for rNav1.4, P04775 for rNav1.2a, P04774 for
rNav1.4 with their rNav1.2a equivarNav1.1, P08104 for rNav1.3, Q14524 for hNav1.5, CAA70364 for rNav1.6, O08562 for rNav1.7). Numbers in
superscript provide the position of the indicated residues in the channel sequence. B, differences in conduc- lents (H599Q/D601S/N602S and
tance-voltage relations of rNav1.4 mutants in the presence of Css4E15A. Open circles designate control and Q657E). Analysis of the eight channel
closed circles the results obtained at various concentrations of Css4E15A (see Table 1). The current-voltage
mutants in the presence of Css4E15A
relations were determined as described in Fig. 2.
revealed a similar negative shift in the
TABLE 2
G/V relations for rNav1.4E592A, rNav1.4H599Q/D601S/N602S,
EC50 for activation of mutant rNav1.4 channels by Css4 mutants
rNav1.4E650A, and rNav1.4Q657E and the unmodified channel. In
EC50 values (M) were determined as described in supplemental Fig. S2. The data
contrast, channel mutations L653A and G658N abolished the
represent the mean S.E. for the independent experiments (number in parentheCss4E15A effect as indicated by the unaffected G/V relations
ses). ND, not determined.
EC50
measured with up to 10 M toxin (Table 1 and Fig. 4). The G/V
rNav1.4
mutant
Css4E15A
Css4F14A/E15A Css4E15A/R27Q Css4E15A/E28R
relations of rNav1.4E1251N and rNav1.4H1257K were affected by
Css4E15A, but with lower potency (EC50 1.91 and 5.3 M,
M
M
M
M
Wild-type 0.88 0.12 (6) 0.93 0.15 (4) 26.2 2.9 (4) 0.98 0.03 (4)
respectively) (Tables 1 and 2 and Fig. 4B). These results suggest
E592A
1.11 0.3 (5) 19.2 2.1 (4)
ND
ND
that Leu-653 and Gly-658, and to a lesser extent Glu-1251 and
E650A
1.26 0.17 (4)
ND
ND
29.8 3.1 (3)
E1251N
1.91 0.17 (4)
ND
2.31 0.3 (4) 23.9 2.7 (3)
His-1257, are involved in the interaction of Css4E15A with
rNav1.4.
Mutant Double Cycle Analysis of Css4 against rNav1.4The
3B). In a concentration of 1 M, Css4F14A/E15A/E28R shifted the
voltage dependence of activation of rNav1.4R666G to V0.5 high activity of Css4F14A/E15A, Css4E15A/E28R, and especially
25 0.7 mV, and less than 15% of the mutant channel pop- Css4F14A/E15A/E28R at the skeletal muscle channel and the lack of
ulation was activated at more negative membrane potentials (Fig. effect at the brain channel indicate that Phe-14 and Glu-28 are
3A). In addition, the steady-state fast inactivation of rNav1.4R666G two major points of difference in the interface between Css4

Nav1.4 Architecture Uncovered by a Specific Activator

OCTOBER 5, 2007 VOLUME 282 NUMBER 40

JOURNAL OF BIOLOGICAL CHEMISTRY

29429

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DISCUSSION
The design of Css4F14A/E15A/E28R,
a specific activator of rNav1.4, was
based on systematic analysis of the
binding of a large collection of Css4
mutants to Navs in rat brain and rat
skeletal muscle membrane preparations and the finding that substitution of Phe-14 and Glu-28 for Ala
markedly decreased activity at the
brain channel without affecting
activity at the muscle channel.
Despite these differences, the substitution of a number of other Css4
residues had a similar impact on the
two channel subtypes, suggesting
FIGURE 5. Schematic presentation of the inter-domain arrangement of the voltage-sensing and pore
modules in rNav1.4. A, schematic Css4 structural model (in ribbon) (23) based on the known structure of the that the toxin recognizes a similar
-toxin Cn2 (90% similarity in sequence) (35) (PDB accession 1cn2) highlighting the three residues shown region on both channels. The simhere to have coupling energies with channel residues derived from three distinct extracellular loops of ilar/non-similar face of toxin interDomains 2 (yellow) and 3 (blue). B, external view of the proposed inter-domain arrangement of the transmembrane segments (S1-S6) in the mammalian Nav based on panel A and following the proposed similarity in action with these channels raised
intra-domain arrangement between NaChBac and Kv1.2 (3, 6). The dashed line illustrates a Css4 projection in its two experimental avenues that
putative bound form to demonstrate the size relations of the toxin and the channel.
could exploit Css4F14A/E15A/E28R.
The first was to assess the therapeuand the two types of Navs. Based on the general conservation of tic potential of such a specific Na activator, and the second was
v
mammalian Navs and the elucidation of a subset of common to examine its interaction with the skeletal muscle channel.
residues involved in the interaction of Css4 with the two chanThe Putative Therapeutic Potential of Css4F14A/E15A/E28R in
nel types, we compared the effects of Css4E15A, Css4F14A/E15A, Neuromuscular DisordersOver thirty mutations in SCN4A,
Css4E15A/E28R, and Css4F14A/E15A/E28R on the activation prop- the gene encoding the skeletal muscle Na , have been linked to
v
erties of the eight rNav1.4 mutants. In addition, we included in neuromuscular disorders such as hypo- and hyperkalemic
E15A/R27Q
this comparative analysis Css4
because of the proximperiodic paralyses, paramyotonia congenital, Nav myotonias,
ity of Arg-27 to Glu-28 and the similar effect of its substitution
and congenital myasthenic syndrome (8). Three of these mutaon both rNav1.2a and rNav1.4 (supplemental Table S1 and
tions were localized to D2/S4 of hNav1.4 (R669H, R672G/H/S,
Table 1) (25). Where a significant change in activity was
and R675G/Q/W) (reviewed in Ref. 8), and one was localized to
obtained (Table 1), the EC50 values were determined, enabling
D3/S4 of hNav1.4 (R1132Q) (14). Skeletal muscle fibers from a
mutant double cycle analysis (Table 2 and supplemental Fig.
patient
heterozygous for R672G displayed depolarization and
S2). The EC50 of Css4F14A/E15A at rNav1.4E592A was 20-fold
weakness in low potassium extracellular solution (15). Both the
higher than its EC50 at the unmodified channel (19.2 2.1
increased inactivation and the impaired voltage dependence of
versus 0.93 0.15 M; Table 2), indicating a coupling energy of
1.65 kcal/mol between F14A of the toxin and E592A of the activation caused by the R672G mutation may contribute to the
channel. Css4E15A/R27Q hardly affected rNav1.4 (EC50 26.2 reduction of Nav performance and reduced membrane excit2.9 M), indicating the importance of Arg-27 for Css4 interac- ability (14). Sternberg et al. (34) reported a very severe hypoPP
tion with its rNav1.4 receptor site (Table 2). Of the eight chan- phenotype in a family carrying the R672G mutation where the
to treatnel mutations, E1251N significantly increased the ability of frequency and severity of attacks increased in response
F14A/E15A/E28R
E15A/R27Q
ment
with
acetazolamide.
The
ability
of
Css4
to
Css4
to produce a negative shift in the conductanceR666G
restore
the
gating
properties
of
rNa
1.4
(Fig.
3),
which
v
voltage relations of the channel (Tables 1 and 2). Indeed, the
R672G
, demonstrates a
potency of Css4E15A/R27Q at rNav1.4E1251N was very similar to mimicked the hypoPP mutation hNav1.4
E15A
putative
therapeutic
potential
when
seeking
a
remedy
to the defecthat of Css4
(Table 2). This indicates that R27Q has a sigF14A/E15A/E28R
tive
SCN4A
gene
product.
The
specificity
of
Css4
for
nificant positive coupling energy with E1251N (G 1.89
E15A/E28R
the
skeletal
muscle
Na
suggests
that
it
merits
investigation
as
a
kcal/mol). Most intriguing were the effects of Css4
v
on
E15A/E28R
possible
treatment
for
hypoPP
and
perhaps
other
neuromuscuthe channel mutants. Whereas the EC50 of Css4
for the
unmodified channel was very similar to that of Css4E15A (0.98 lar disorders with symptoms of reduced muscle excitability.
Derivation of Channel Architecture from Css4-rNav1.4
0.03 M), it increased prominently for rNav1.4E650A and
E1251N
Mutant
Double Cycle AnalysisThe interaction of Css4 and
rNav1.4
(29.8 3.1 and 23.9 2.7 M, respectively)
rNa
1.4
was
examined by mutant double cycle analysis, focus(Tables 1 and 2 and Fig. 4B). Hence, a single Css4 residue (Gluv
28) exhibits negative coupling energy with two channel residues ing on residues employed in the design of the selective activator
(G 1.8 and 1.41 kcal/mol for E650A and E1251N, and on rNav1.4 channel residues whose equivalents were either
respectively) positioned on two distinct extracellular loops in proposed to be involved in the interaction of Css4 with rNav1.2a
Domains 2 and 3 (D2/S3-S4 and D3/SS2-S6).
(20) or whose substitution was shown to affect the interaction

Nav1.4 Architecture Uncovered by a Specific Activator

AcknowledgmentWe thank Prof. F. Frolow, Tel Aviv University, for

help with the illustration of the toxin-channel interaction.

29430 JOURNAL OF BIOLOGICAL CHEMISTRY

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of the toxin Tz1 with rNav1.4 (22). Four conserved residues in

the external loops of rNav1.2a have been implicated in the interaction with Css4: Glu-779 in D2/S1-S2 and Glu837, Leu-840,
and Gly-845 in D2/S3-S4 (Fig. 4A) (20). The decrease in the
ability of Css4E15A to modulate channel activation following
substitutions of L653A and G658N in rNav1.4 (equivalent positions of Leu-840 and Gly-845 in rNav1.2a) (Table 1 and Fig. 4A)
suggests that these residues belong to a conserved region of
receptor site 4. However, the lack of effect of substitutions
E592A and E650A in rNav1.4 (equivalent positions in rNav1.2a
are Glu-779 and Glu-837) on Css4E15A action indicates that
despite their conservation in all mammalian Navs these residues do not belong to the common receptor for scorpion -toxins on Navs (Table 1 and Fig. 4A).
Overall, the substantial variations in the receptor site for
Css4 in rNav1.2a and rNav1.4 are consistent with the results of
binding assays of Css4 mutants on rat brain and muscle Navs
and are further demonstrated by the difference in the face of
interaction between the two channels and the triple mutant
Css4F14A/E15A/E28R (supplemental Table S1). In light of the general conservation of mammalian Navs, the high specificity of the
triple Css4 mutant for Nav1.4 suggests that the toxin residues
Phe-14 and Glu-28 encounter a different face upon binding to
rNav1.2a versus rNav1.4. This prompted us to examine by
mutant cycle analysis Css4-rNav1.4 interacting pairs. We
focused on the toxin residues Phe-14 and Arg-28, whose substitution abolished the activity toward rNav1.2a, but not
rNav1.4, and Arg-27, whose substitution affected both channel
types (Tables 1 and 2 and Fig. 2). In the channel we selected
those residues whose substitution was shown to influence the
effect of scorpion -toxins (20, 22). The significant coupling
energies obtained between F14A (toxin) and E592A at
D2/S1-S2 (channel), as well as E28R (toxin) and the two channel
residues E650A at D2/S3-S4 and E1251N at D3/SS2-S6 (Table
2), along with the three-dimensional model of Css4 (Fig. 5) (25,
35) enabled to estimate the distances between the toxin channel
interacting pairs. As the distance between C of Phe-14 and C
of Glu-28 is 6 8 , Glu-592 of D2/S1-S2 is likely to reside 10
from Glu-650 of D2/S3-S4. Because E28R of the toxin demonstrated a negative energy of interaction with both E650A of
D2/S3-S4 and E1251N of D3/SS2-S6, we conclude that the two
channel residues are very close to one another (Fig. 5). This
conclusion is further corroborated by the high positive coupling energy between R27Q of the toxin and E1251N of the
channel (Tables 1 and 2). Based on these data and in the absence
of a three-dimensional structure of the Nav, we suggest that
loop S3-S4 of the voltage sensor module in Domain 2 is in very
close proximity with loop SS2-S6 of the pore module in Domain 3.
Although substantiation of this suggestion requires resolution of
the channel three-dimensional structure, the proposed architecture resembles that reported for Kvs, where the voltage-sensing
module of each domain is in close proximity to the pore module of
the adjacent domain, in a clockwise orientation (Fig. 5) (3).