The polypeptide neurotoxin anthopleurin B (ApB)
isolated from the venom of the sea anemone Anthopleura
xanthogrammica is one of a family of toxins that bind to the
extracellular face of voltage-dependent sodium channels and
retard channel inactivation. Because most regions of the sodium channel
known to contribute to inactivation are located intracellularly or
within the membrane bilayer, identification of the toxin/channel
binding site is of obvious interest. Recently, mutation of a glutamic
acid residue on the extracellular face of the fourth domain of the rat
neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel
binding (Rogers, J. C., Qu, Y. S., Tanada, T. N.,
Scheuer, T., and Catterall, W. A. (1996) J. Biol.
Chem. 271, 15950-15962). A negative charge at this position is
highly conserved between mammalian sodium channel isoforms. We have
constructed mutations of the corresponding residue (Asp-1612) in the
rat cardiac channel isoform (rH1) and shown that the lowered affinity
occurs primarily through an increase in the toxin/channel dissociation
rate koff. Further, we have used thermodynamic
mutant cycle analysis to demonstrate a specific interaction between
this anionic amino acid and Lys-37 of ApB (
G = 1.5 kcal/mol), a residue that is conserved among many sea anemone
toxins. Reversal of the charge at Asp-1612, as in the mutant D1612R,
also affects channel inactivation independent of toxin (
14 mV shift
in channel availability). Binding of the toxin to Asp-1612 may
therefore contribute both to toxin/channel affinity and to transduction
of the effects of the toxin on channel kinetics.
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INTRODUCTION |
Members of a structurally diverse family of toxins isolated from
several species of sea anemone and scorpion have proven useful tools in
the functional characterization of mammalian voltage-gated sodium (Na)
channels (1, 2). These toxins bind to the extracellular face of the
channel at a locus termed site-3 (3) and selectively delay current
inactivation (4-6), principally by inhibiting transitions of the
channel from the open to the inactivated state (7-9). Several of the
sea anemone toxins discriminate between different mammalian isoforms of
the Na channel, generally binding to the cardiac isoform with highest
affinity (10). Because these toxins are only active when applied
extracellularly (5, 6) and because most regions of the channel known to
affect inactivation are located intracellularly or within the plasma
membrane (11-14), it is of particular interest to characterize the
binding interface between toxin and channel.
Several regions of the channel have been implicated in toxin binding.
Studies involving covalent attachment of scorpion toxin derivatives
(15) and protection against such labeling by site-specific antibodies
(16) have implicated regions in the first channel domain in toxin
binding, while other studies involving antibodies (16) and chimeric
channels (17) have pointed to regions in domain 4. Most recently,
mutation of a negative amino acid at the outer end of the third helix
in the fourth domain of rat brain sodium channels (Glu-1613) was shown
to inhibit the binding of the sea anemone toxin ATX-II (from
Anemonia sulcata) by 80-fold (18).
The sea anemone toxin anthopleurin B
(ApB)1 was originally
isolated from the Pacific coast sea anemone Anthopleura
xanthogrammica (19, 20) and subsequently cloned and expressed
heterologously (21). It has seven positive amino acid residues (22),
and the ability of a subset of those to affect the binding affinity of
the toxin (23-25) suggested the possibility of an electrostatic interaction between toxin and channel. We therefore attempted to
determine which of these cationic toxin residues mediate the decrease
in affinity observed with the channel mutation at the end of domain IV,
helix 3 (18). Specifically, we constructed a series of mutations of
residues on ApB and used the formalism of thermodynamic mutant cycle
analysis (26, 27) to assess the degree to which these toxin mutations
interacted with the mutation on the channel. Taking advantage of the
higher affinity of ApB for cardiac sodium channels than for neuronal
channels, we performed our experiments in cells transfected with the
rat cardiac channel isoform rH1.
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MATERIALS AND METHODS |
Molecular Biology--
The rat heart sodium channel cDNA
(rH1) (28, 29) subcloned into the pSP64t vector was generously provided
by R. Kallen (University of Pennsylvania). The aspartic acid at amino
acid 1612 (corresponding to position 1613 in rBr2a) was mutated to asparagine (D1612N) or arginine (D1612R) using polymerase chain reaction (PCR) in a 4-primer strategy (30). Sequences in this region
were as follows,
where the capitalized residues denote the position of Glu-1613
(rBr2a) and Asp-1612 (rH1) and asterisks denote the third and fourth
helices of channel repeat IV (see Ref. 31). PCR primers included the
desired mutations and an additional mutation to introduce a silent
KpnI restriction site, which did not alter the amino acid
sequence but facilitated identification of mutated PCR products. The
final PCR products spanned unique BstEII (base pair 4632) and BspDI (base pair 5069) endonuclease restriction sites in
rH1 and were subsequently used in the reconstruction of the full-length rH1 cDNA in pSP64t. The entire BstEII to
BspDI PCR-generated region found in the full-length
construct was completely sequenced by dideoxy methods (32) to confirm
the presence of the desired mutations and the absence of spurious
PCR-generated errors. Expression was first tested in Xenopus
oocytes, and the entire coding region of rH1 was subsequently shuttled
into the HindIII site in the polylinker of pREP9
(Invitrogen, Carlsbad, CA) using HindIII (in pSP64t) to
HindIII (base pair 6371 in rH1) endonuclease restriction sites.
Cell Culture--
Channels from the rH1-pREP9 constructs were
expressed in tsA201 cells (33) and maintained as described previously
(17). Two to three days prior to recording, cells were seeded onto
60-mm culture dishes and transfected with 10 µg of plasmid pREP9-rH1 using a calcium phosphate coprecipitate. Cells were cotransfected with
pHook1, and transfectants were visualized with the Capture-Tec bead
system (Invitrogen) using supplied protocols. Approximately 30-50% of
cells bound beads; virtually all cells binding two or more beads
expressed Na+ current (INa).
Electrophysiological Solutions--
Pipette solution contained
(in mM) 140 Cs+, 10 Na+, 100 F
, 50 Cl
, 10 HEPES, with pH adjusted to 7.3 with CsOH. Bath solution contained (in mM) 140 Na+, 2 Ca2+, 144 Cl
, 10 HEPES,
with pH adjusted to 7.3 with NaOH. Because toxin in solution tends to
adhere to glassware and tubing, bath solutions containing toxin were
supplemented with 5 mg/ml bovine serum albumin (Sigma) (17). Toxins
ApB, ApA, and ApB mutants were produced in Escherichia coli
strain BL21(DE3) transformed with the appropriate version of the
plasmid pKB-13 as described previously (21, 25, 34) and characterized
by amino acid analysis and matrix-assisted laser
desorption/ionization-time of flight mass spectrometry. Circular
dichroism spectra of the toxin derivatives used in the present study to
confirm proper folding of toxins were determined in a Jasco 710 spectropolarimeter (Easton, MD) equilibrated with camphor
d10 sulfonic acid, and the spectra deconvoluted
using the standard error of the estimate program supplied by the
manufacturer.
Recording Techniques--
Recording protocols were generated
using Clampex 6.0.2 (Axon Instruments, Foster City, CA) running on a
Pentium-based microcomputer and were imposed through a 12-bit DAC
controlling an Axopatch 200B amplifier (Axon). The recording chamber
was maintained at 16 °C with a Sensortek TS-4 controller (Clifton,
NJ). Currents were 4-pole Bessel filtered at 5 kHz and digitized at
12-bit resolution at 100 kHz.
Recordings were made in the whole-cell patch clamp configuration with
typical pipette resistances of 0.8-1.5 M
. Because the binding of
these toxins is state-dependent, with significantly lower
affinity for inactivated states than for resting states (35-37), we
held the cells at a sufficiently negative holding potential (
150 mV)
to maintain complete channel availability during the duration of the
experiment. Our experiments therefore probed the interactions of these
toxins primarily with the closed conformation of the channel.
Data Analysis--
Analysis and fitting were performed with
locally written programs (GRB, DAH) running under Matlab 5.0 (The
MathWorks, Natick MA). Traces were leak corrected based on the
resistance calculated from the current at holding potential. The
capacity transient was removed through subtraction of summed
subthreshold pulses, and traces were digitally refiltered at 5 kHz
using a zero-phase fitting algorithm.
When the rates of current modification or unmodification by toxin were
too fast to resolve with pulse protocols, toxin/channel affinities were
calculated using the currents produced by modifying channels with
subsaturating concentrations of toxin. We assumed that partially
modified INa consisted of the sum of a quickly decaying component arising from unmodified channels and a slowly decaying phase arising from toxin-modified channels. The current at
late times after the start of depolarization was, therefore, assumed to
be proportional to the total number of channels modified. Accordingly,
we averaged the current over a 7.6-8.0-ms window from depolarization
onset (I7.6, low). Because peak currents increase during toxin modification, we normalized this late current by
the initial value of peak current before toxin modification (Ipeak, low). This ratio was then divided by a
similarly calculated ratio for a saturating dose of a higher affinity
toxin from another cell
(I7.6, high/Ipeak, high),
yielding the fraction of current modified
(fmod). That is as follows.
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(Eq. 1)
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Affinity was then calculated from the Langmuir adsorption
isotherm.
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(Eq. 2)
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Interactions between toxin and channel residues were calculated
using the formalism of thermodynamic mutant cycle analysis (26, 27). In
brief, wild-type and mutant forms of toxin and channel were used to
calculate a coupling coefficient
,
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(Eq. 3)
|
where, for example, KD(mut, wt) denotes the
affinity of mutant channel for wild-type toxin. The energy of
interaction between the two residues was then calculated as
follows.
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(Eq. 4)
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Because affinities were related to coupling energies
logarithmically, and because affinities and rate constants both varied over a wide range of values, we chose to calculate errors and significance statistics based on the natural logarithms of the data.
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RESULTS |
Kinetics of Mutant Channels--
We introduced mutations into rH1
at the position identified by Rogers and colleagues (18) in rat brain
channels (rBrIIa). Channels rH1 D1612N and D1612R displayed currents
similar to those of the wild-type channel (Fig.
1A). The mutations produced
little change in voltage-dependence of conductance (Fig. 1B,
left); the charge neutralization mutant D1612N was very
slightly but significantly shifted in a depolarizing direction
(p
0.02, Student's t-test), while the
halfpoint of the charge reversal mutation was unchanged. Halfpoints of
the conductance curve were as follows: wild-type rH1,
41 ± 1 (n = 14); D1612N,
38 ± 2 (n = 13); D1612R,
43 ± 2 (n = 9). Cells with
conductance slope factors <6.0 mV were deemed to be inadequately
voltage controlled and were excluded from any further study (38). On
the other hand, the charge reversal mutation D1612R displayed a
steady-state availability curve 14 mV negative to that of wild-type
channels (p
0.0001), whereas that of D1612N was
similar to the wild-type channel (Fig. 1B,
right). Halfpoints of availability were: wild-type rH1,
100 ± 2 (n = 22); D1612N,
101 ± 2 (n = 14); D1612R,
114 ± 2 (n = 9). Paralleling the shift in availability, the kinetics of recovery
from inactivation of D1612R were shifted in a hyperpolarizing direction
by
16 ± 3 mV across a range of recovery potentials with respect
to rH1 (data not shown).

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Fig. 1.
Kinetic characterization of wild-type and
mutant rH1 channels. tsA201 cells were transfected with wild-type
rH1 ( ); rH1 D1612N ( ); and rH1 D1612R DNA ( ). Horizontal
scale bar, 5 ms. A, raw currents evoked by depolarizing
pulses from a holding potential of 150 mV up to +20 mV at 10 mV
intervals. Traces were leak- and capacity-corrected as described under
"Materials and Methods." Vertical scale bars, 500 pA.
B, normalized steady-state availability (left
curves) and conductance (right curves) relations of
wild-type and mutant rH1 channels. Insets show voltage
protocols. Steady-state availability curves were fit with the Boltzmann
equation, and current/voltage relations were fit with the product of
the Boltzmann equation and Ohm's law. Average values of fit parameters and n values are given in the text. C, currents
evoked by depolarizing pulses to 10 mV before (top traces)
and after (bottom traces) modification to steady-state with
100 nM ApB. Traces were leak- and capacity-corrected as
described under "Materials and Methods."
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Neutralization (D1612N) or reversal (D1612R) of Asp-1612 decreased the
affinity for ApB, consistent with results in the neuronal channel
isoform (18). For example, 100 nM ApB modified wild-type rH1 channels to completion but caused only fractional modification of
current from the charge reversal mutation D1612R (Fig.
1C).
Calculation of Toxin/Channel Interaction--
The energies of
interaction of the positive residues of ApB with channel residue
Asp-1612 were calculated through thermodynamic mutant cycle analysis
(26, 27). In short, the effects of point mutations of ApB on
toxin/channel affinity were tested both in the context of wild-type rH1
channels and in the context of channels mutated at Asp-1612.
Differences in the effects of the toxin mutation were quantified as
coupling coefficients
, which are related logarithmically to the
energy of interaction between the particular toxin and channel residues
(see "Materials and Methods").
Because association rates of these toxins are 3 orders of magnitude
below those predicted by diffusion, we were usually able to calculate
affinities using the rate constants of toxin/channel association and
dissociation. Once stable whole-cell access was achieved, cells were
exposed to and removed from a toxin-containing chamber. During wash-in
and wash-out, modification was monitored by 11-ms depolarizations to
10 mV at
1 Hz, of which a current window of 7.6-8.0 ms after the
onset of each pulse was averaged and plotted (Fig.
2A). For most combinations of
toxin and channel, these relations were well resolved by single
exponentials, yielding first order rate constants of association,
kmod, and dissociation, koff, respectively (Fig. 2A). From
these values, the true second-order association rates,
kon, and the affinities, KD,
were calculated as follows,
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(Eq. 5)
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where T denotes toxin concentration, and
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(Eq. 6)
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In most cases, toxin-channel affinities were sufficiently high
that modification of INa was produced at
measurable values of kmod. However, in the cases
where toxin/channel affinity was quite low (specifically, the binding
of all toxins to rH1 D1612R and the binding of ApA to D1612N), a
concentration of toxin sufficient to cause appreciable modification
produced values of kmod too high to accurately
resolve the modification process. In those cases, affinity was
calculated from the currents of cells modified to steady-state with
subsaturating toxin concentrations, as described under "Materials and
Methods." We verified this technique by calculating the binding
affinity of toxin ApB H39A to rH1 D1612N channels both kinetically and
by steady-state measurements. The kinetically calculated
KD, 26 nM (25-27 nm, n = 4), agreed well with the equilibrium measurement of 20 nM
(13-31 nm, n = 5).

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Fig. 2.
Affinities and interactions of toxins with
residue Asp-1612. A, sample affinity calculation. A cell
expressing rH1 D1612N channels was modified to steady-state with 100 nM ApB H39A toxin. Average current over a window 7.6-8.0
ms after depolarization onsets (dotted lines in
insets) is shown during treatment with toxin ( ,
left) and subsequent wash-out ( , right). Note
the change in scale along the time axis. Smooth curves show
fits to data by single exponentials with rate constants
kmod and koff as shown. Insets show INa before toxin
modification (1), at steady-state (2), before
unmodification (3), and after unmodification (4). Vertical scale bar, 500 pA; horizontal scale bar,
2 ms. Cell 97424-5, Rm = 1.5 G ,
Cm = 23 picofarad. B, values of
KD calculated from association and dissociation
kinetics, except for the binding of ApA to rH1 D1612N, which was
obtained from steady-state measurements. Note logarithmic scale.
Black, affinities to rH1; white, affinities to
rH1 D1612N. C, coupling coefficients (left axis,
logarithmic scale) and energies of interaction (right axis,
linear scale) are shown for channel residue Asp-1612 and the indicated
toxin residues. (In keeping with convention (26), any values of < 1 are shown as reciprocals.) n values are shown in Table
I.
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Toxin Interactions with D1612N--
Including its
-amino group,
ApB possesses six fully positive charges and two histidines (22), any
of which might be a candidate for binding to the negative channel
residue Asp-1612. Residues Arg-14, His-34, His-39, and Lys-48 have
produced only small effects on toxin affinity when neutralized (25, 39)
and so were relatively poor candidates for interacting with Asp-1612;
similarly, short N-terminal extensions of the toxin have no measurable
effect on its affinity (21, 40). We tested toxin ApB H39A as a
representative of this group. Residues Arg-12 and Lys-49 are known to
have a significant and cooperative effect on toxin affinity (23) but are not conserved among all anemone toxins; in particular, they are
neutral in ATX-II, the toxin tested by Rogers and colleagues (18). We
tested these residues by using toxin ApA, which has neutral residues at
both positions (in addition to several more conservative differences
which have been shown to have only minor effects on toxin affinity)
(22, 40). The remaining residue, Lys-37, significantly decreased the
affinity of ApB when neutralized (25) and is also conserved among a
number of anemone toxins. It was, therefore, a strong candidate for the
interaction and was tested with toxin ApB K37A. As a positive control,
we also tested a toxin mutated at a hydrophobic position, toxin ApB
W45F. This toxin has lower affinity for cardiac channels than does
wild-type ApB (34) but would not be expected to interact with an
anionic channel residue.
Values of koff and kon
for wild-type channels and for rH1 D1612N are shown in Table
I. The decrease in affinity with channel mutation D1612N arose primarily from an increase in
koff, as did the difference in affinity between
ApB and ApA, which was consistent with previous results (23). The
effects of toxin mutation K37A, however, arose from both a decrease of
kon and an increase in koff. These kinetic values were used to
calculate toxin/channel affinities (Fig. 2B), which were
then translated into coupling coefficients (
) and energies of
interaction (Fig. 2C). Of the tested toxin mutations, only
K37A interacted with channel mutation D1612N with an energy
significantly different from zero (
G = 1.5 kcal/mol). We conclude that toxin residue Lys-37 interacts positively
with channel residue Asp-1612.
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Table I
Kinetic rate constants of toxin binding to rH1 and rH1 D1612N
Rate constants were calculated from first-order modification and
unmodification rates as described in text.
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Toxin Interactions with D1612R--
Having demonstrated an
attraction between cationic toxin residue Lys-37 and anionic channel
residue Asp-1612, we attempted to preserve this interaction by
transposing the charges of the two residues. To this end, we
constructed the charge-reversed toxin ApB K37D and the charge-reversed
channel rH1 D1612R. Unfortunately, circular dichroism revealed that ApB
K37D possessed a significantly less ordered secondary structure than
that of wild-type ApB or the neutralization mutant ApB K37A (25) (Fig.
3).

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Fig. 3.
Circular dichroism spectra of wild-type ApB
(line), ApB K37D ( ), and ApB K37A ( ). Spectra
were measured at room temperature and neutral pH in cylindrical quartz
cells at protein concentrations of 100-200 µg/ml and deconvoluted by
least squares comparison to a data base containing proteins of known structure (cytochrome C, ribonuclease A, myoglobin, hemoglobin, papain,
-chymotrypsinogen, trypsin, lysozyme, and alcohol dehydrogenase). Calculated secondary structure was as follows: ApB, 0.8% -helix, 52.3% -sheet, 21.0% -turn; ApB K37A, 0.0% -helix, 50.9%
-sheet, 18.8% -turn; ApB K37D, 1.0% -helix, 17.1%
-sheet. 8.2% -turn (ApB and ApB K37A, 25).
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Accordingly, ApB K37D bound to both wild-type rH1 channels and to rH1
D1612R with strikingly low affinity (see Table
II). Although the calculated interaction
was large (
G = 2.2 kcal/mol), we interpret this
"interaction" to arise from aberrant folding of the toxin rather
than from the targeted disruption of an ionic interaction between
Lys-37 on the toxin and Asp-1612 on the channel. Somewhat surprisingly,
the energy of interaction of the neutralized toxin, ApB K37A, with the
charge-reversed channel, rH1 D1612R, was only 0.9 kcal/mol, a lower
value than that observed when both the channel and toxin residues were
neutralized.
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Table II
Affinities of toxins to rH1 and rH1 D1612R
Affinities were determined by equilibrium modification as described
under "Materials and Methods."
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DISCUSSION |
We have isolated a specific interaction involved in the binding of
the site-3 toxin anthopleurin B to the rat cardiac sodium channel.
Channel residue Asp-1612 and toxin residue Lys-37 display an energy of
interaction of 
G = 1.5 kcal/mol. This coupling energy is less than that expected for a hydrogen bond to a charged residue (41) but is well within the range predicted for electrostatic interactions (41). For example, Schreiber and Fersht (42) reported
values ranging from a noise level of 0.4 kcal/mol up to values typical
of H-bonds. Indeed, while neutralization of Lys-37 in the context of a
wild-type channel decreased affinity by 15-fold, the mutant channel
D1612N was almost insensitive to a charge at toxin position 37. Thus
all of the effects of neutralizing toxin residue Lys-37 on the affinity
of the toxin are accountable through the actions of channel residue
Asp-1612. A comparison of the sequences of 15 homologous toxins
isolated from sea anemone revealed all toxins but one, ATX-I from
A. sulcata (43), to have a positive residue at or adjacent
to position 37. Interestingly, toxin ATX-I is ineffective when applied
to vertebrate sodium channels at 100 µM (5). Further, an
anionic residue at channel position 1612 is highly conserved between
mammalian sodium channel isoforms (31). These data strongly support the
hypothesis that the interaction between Lys-37 and Asp-1612 comprises a
conserved component of binding site-3 in mammalian channels.
Site-3 toxins act to delay sodium channel inactivation (4-6). Because
the extracellular face of the channel is not typically thought to be
associated with the inactivation process, the manner by which these
toxins produce their kinetic effect remains unclear. In light of this
ambiguity, it is interesting that reversal of the charge at channel
residue Asp-1612 produced a shift in the voltage dependence of
steady-state availability. Since perturbation of this residue affects
inactivation gating, it is possible that Asp-1612 participates in the
control of both the affinity and the kinetic effects of toxin
binding.
At present, little is known of the structure of the extracellular face
of the sodium channel outside of the immediate region of the pore
vestibule. The interaction between Asp-1612 and toxin residue Lys-37
provides a defined interaction between the channel and a molecule with
a known three-dimensional structure (44). The determination of more
such interactions should therefore increase our insight into the
structure and function of the exterior of the channel.
We thank Dr. Roland Kallen for the kind gift
of the rH1 clone and Ian Glaaser and Al Combs for help with molecular
biology.