Distinct Sites Regulating Grayanotoxin Binding and Unbinding to
D4S6 of Nav1.4 Sodium Channel as Revealed by Improved
Estimation of Toxin Sensitivity*
Hiroshi
Maejima
,
Eiji
Kinoshita§,
Issei
Seyama¶, and
Kaoru
Yamaoka§
From the
Institute of Health Sciences and the
§ Department of Physiology, School of Medicine, Hiroshima
University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551 and the
¶ Faculty of Human Life Science, Hiroshima Jogakuin University,
Ushita Higashi 4-13-1, Higashi-ku, Hiroshima 732-0063, Japan
Received for publication, November 29, 2002, and in revised form, January 10, 2003
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ABSTRACT |
Grayanotoxin (GTX) exerts selective effects on
voltage-dependent sodium channels by eliminating fast
sodium inactivation and causing a hyperpolarizing shift in voltage
dependence of channel activation. In this study, we adopted a newly
developed protocol that provides independent estimates of the binding
and unbinding rate constants of GTX (kon and
koff) to GTX sites on the sodium channel
protein, important in the molecular analysis of channel modification.
Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276)
by means of site-directed mutagenesis; the results suggested that the
GTX receptor consists of the S6 transmembrane segments of four
homologous domains facing the ion-conducting pore. We systematically
introduced at two sites in D4S6 (Nav1.4-Phe-1579 and
Nav1.4-Tyr-1586) amino acid substituents with residues
containing hydrophobic, aromatic, charged, or polar groups. Generally,
substitutions at Phe-1579 increased both kon
and koff, resulting in no prominent change in
dissociation constant (Kd). It seems that the smaller the molecular size of the residue at
Nav1.4-Phe-1579, the larger the rates of
kon and koff,
indicating that this site acts as a gate regulating access of toxin
molecules to a receptor site. Substitutions at Tyr-1586 selectively
increased koff but had virtually no effect on
kon, thus causing a drastic increase in
Kd. At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to
GTX. These results suggest that the residue at position Tyr-1586 has a
more critical role in mediating GTX binding than the one at position
Phe-1579. Here, we propose that the affinity of GTX to
Nav1.4 sodium channels might be regulated by two residues
(Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.
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INTRODUCTION |
Biological toxins that act selectively on the
voltage-dependent sodium channels have been used to
elucidate molecular mechanisms of sodium channel gating processes.
Batrachotoxin (BTX),1
grayanotoxin (GTX), veratridine, and aconitine, classified as Site 2 toxins (1), are endowed with some characteristics in common
(2-5); 1) they bind to the sodium channel in its open state, 2) the
modified sodium channel lacks the inactivation process, and 3) the
activation potential of the modified sodium channel is shifted in the
direction of hyperpolarization. Among the well characterized toxins,
GTX has distinct advantages in that the relevant interaction sites on
the GTX molecule with the sodium channel are known as a result of
extensive analyses of its structure-activity relationship (6-8). The
methyl group of the
-surface of the molecule at C-10 and the
hydroxyl groups of its
-surface at C-3, C-5, and C-6 are essential
to the pharmacological action of GTX and the hydrophobic
microenvironment of the
-surface, of which C-14S is most
critical, greatly contribute to increased GTX potency (see Fig.
2A). Moreover, the GTX action has a unique dependence on the
state of the sodium channel; although BTX binds to and dissociates from
the channel in its open state (9), GTX binds only to the open channel
and dissociates from the closed channel (10).
The main structural component of sodium channels is the pore-forming
-subunit, whose molecular mass is more than 250 kDa. The
-subunit
consists of four homologous domains (D1-D4), each containing six
-helical transmembrane segments (S1-S6) (11, 12). The regions
between S5 and S6 in each of the four domains form pore loops that dip
into the membrane to create a narrow selectivity filter at the external
end of the ion-conducting pore. The remainder of the pore is formed by
each S6 segment of the four domains in close apposition to one another
(13, 14). Recent site-directed mutagenesis studies show that specific
amino acid residues within each of the four S6 segments are important determinants of action of Site 2 toxins (15-23). Previously, we reported that D1S6 and D4S6 segments (but not D2S6 and D3S6) are required for GTX binding to the sodium channel (19-21). Unexplored GTX
sites in D2 and D3 have been examined in this study. Within these
segments, a number of putative binding sites on the rat skeletal muscle
sodium channel isoform, Nav1.4 (Ile-433, Asn-434, Leu-437, Ile-1575, Phe-1579, and Tyr-1586), may interact with the
-surface of the GTX molecule, whose pharmacological action is
effected within a hydrophobic microenvironment (8). Residue Tyr-1586 in
D4S6 has been found to provide a unique binding site for GTX, which
excludes BTX and veratridine. Moreover, we have attributed the
difference in GTX sensitivity of the sodium channel isoforms,
Nav1.4, and the rat cardiac channel, Nav1.5, to
a critical residue (Ser-251) in the intracellular linker of D1S4-S5
(19). Kimura et al. (19) deduced particular GTX binding
sites by making several chimeras targeting the heterologous amino acid
residues of Nav1.4 and Nav1.5; these chimeras
showed reduced sensitivity to GTX. Most recently, Maejima
et al. (24) found novel binding sites both in D1S4-S5 and
D4S4-S5 linkers of Nav1.4 and Nav1.5 through an
alanine-scanning method.
Recently, Yuki et al. (10) developed a novel method to
evaluate the sensitivity of sodium channels to GTX; this method
independently determines the binding and unbinding rate constant
(kon and koff) of GTX to
sodium channels. The sensitivity of sodium channels to GTX is indicated
by the dissociation constant (Kd), i.e.
the ratio of koff to kon.
Here, using this new technique, we reevaluated already-known GTX sites,
hoping to uncover novel roles of these sites in GTX binding. At the
sites in D4S6, we made systematic amino acid substitutions to further
clarify their roles in more detail.
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EXPERIMENTAL PROCEDURES |
Construction of Point Mutants of the Rat Skeletal Muscle Sodium
Channel--
Point mutants of the sodium channel were constructed
using the cDNA clone encoding the rat skeletal muscle sodium
channel
-subunit, Nav1.4 (25). To construct the mutants
by substitutions with various amino acids, we followed a PCR-based
site-directed mutagenesis method as described previously (19-21, 24,
26). All of the resulting mutants were confirmed with restriction
mapping and sequencing using an ABI PRISMTM 310 Genetic
Analyzer (Applied Biosystems, Foster City, CA).
Transient Transfection and Cell Culture--
The constructed
point-mutated cDNA clones were inserted into a mammalian expression
vector pCI-neo (Promega Corp., Madison, WI) or pcDNA3.1
(Invitrogen) and were then transiently co-transfected with CD8 cDNA
into HEK 293 (human embryonic kidney) cells using the SuperFect
transfection reagent (Qiagen, Hilden, Germany). The cells were grown to
50% confluence in Dulbecco's modified Eagle's medium (Invitrogen)
containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 30 units/ml penicillin G (Meiji Corp., Tokyo, Japan), and 30 µg/ml
streptomycin (Meiji Corp.) in a humidified atmosphere of 5%
CO2 and 95% air at 37 °C. The transfected cells were
used for electrophysiological experiments as late as 3-4 days after
being replated in 35-mm tissue culture dishes. Transfection-positive
cells were identified by Immunobeads (CD8-Dynabeads, Dynal, Oslo,
Norway) before Na+ current (INa) recording.
Electrophysiological Recording--
Macroscopic
INa from the transfected cells was measured
using the whole-cell patch clamp method. The bath solution contained 70 mM NaCl, 67 mM
N-methyl-D-glucamine, 1 mM
CaCl2, 1.5 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.4. The pipette
solution contained 70 mM CsF, 60 mM CsCl, 12 mM NaF, 5 mM EGTA, and 5 mM
HEPES, pH 7.4. To assess the effects of GTX on whole-cell
INa, 300 µM GTX I (a GTX analogue)
was added to the pipette solution because GTX is known to act
intracellularly (27), and this concentration is sufficient to evaluate
sensitivity of the mutated channels to GTX (19).
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RESULTS |
Comparison of the Sensitivity to GTX among Wild Type
Nav1.4 and Its Mutant Channels--
Because GTX binds to
sodium channels exclusively in their open state (10, 19-21, 24, 28),
repetitive depolarizing pulses were applied to induce GTX modification.
After these repetitive conditioning pulses, test pulses to various
membrane potentials induced a characteristic sustained current (Fig.
1, A2, B2, and C2). Without repetitive prepulses, the majority of channels
were unmodified by GTX so that the recorded currents in the presence of
300 µM GTX (in the internal medium) exhibited the typical
behavior of unmodified channels. However, closer examination revealed a residual non-inactivating component, indicating that a very small fraction of the channels was modified during the test pulse itself without repetitive preconditioning pulses (Fig. 1, A1,
B1, and C1).

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Fig. 1.
GTX modification of sodium channels.
Differences in sensitivity to GTX I between wild type
Nav1.4 and mutants (F1579A, Y1586A, and N784K).
A-D, INa families and I-V relations
for unmodified peak INa and for GTX-modified
steady-state INa in wild type Nav1.4
(A), mutant F1579A (B), Y1586A (C),
and N784K (D). Control currents (unmodified currents) were
taken in the presence of intracellular GTX. The currents were scarcely
modified without preconditioning pulses even in the presence of 300 µM GTX, as shown in panels A1, B1,
and C1. Modified currents (A2, B2, and
C2) were induced by a train of 100 conditioning prepulses
(pulse potential = 20 mV; pulse duration = 6 ms, interpulse
interval = 100 ms; holding potential = 120 mV). I-V
relationships (A3, B3, C3, and
D3) of unmodified and modified currents were obtained using
160-ms test pulses to a variable potential ( 140 mV to +50 mV in 10-mV
steps); unmodified currents (filled squares) were measured
as peak INa, and modified currents (open
circles) were measured as the steady-state current at the end of
the pulse.
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To permit comparison of data from different cells, we standardized the
currents by computing their maximum chord conductance from
current-voltage curves (19-21, 24, 28). The I-V relationships for
unmodified INa through wild type
Nav1.4 channels and its mutant forms2 (Nav1.4-F1579A
and Nav1.4-Y1586A) are given in Fig. 1, A3,
B3, and C3. The continuous line was fitted to
peak INa at membrane potentials from 0 to +60
mV, and the chord conductance was estimated from the slope. For
GTX-modified currents, application of 100 conditioning pulses evoked an
entirely different family of sustained currents through modified sodium
channels (Fig. 1, A2, B2, and C2).
Modified INa at the end of a 160-ms test pulse
was plotted against the membrane potential, and the chord conductance
of the modified channel population was estimated from the slope of the I-V relationship between
50 and +50 mV (dashed line in
Fig. 1, A3, B3, and C3). We routinely
used the ratio of maximal chord conductances of GTX-modified to
unmodified channels as a measure of channel sensitivity to GTX
modification, which is referred to as the relative chord conductance
(RCC). Our previously reported RCC values for wild type
Nav1.4 and various mutant channels have been provided for
comparison with present results in Table I. As for the F1579A
mutant, our previously reported RCC value of 0.75 (20) was not confirmed in the present study, which yielded a value
of only 0.22.
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Table I
RCC, kon, koff, and Kd in wild type
Nav1.4 and its point mutants
NA means that the GTX modification was so small that it was not
available to measure relative chord conductance, kon
or koff. NE means functional channels were not
expressed in these mutants. Dash denotes data are not available because
experiments were not done in the mutant and exhibited the same value of
RCC as wild type or no functional mutants.
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Novel Binding Sites of GTX in Transmembrane Segment D2S6 and D3S6
of Nav1.4 Sodium Channel--
In our previous studies, it
was confirmed that Ile-433, Asn-434, and Leu-437 in D1S6 and Ile-1575,
Phe-1579, and Tyr-1586 in D4S6 of sodium channels were critical sites
for GTX modification (19-21). Recently, it was reported that Asn-784
and Leu-788 in D2S6 and Ser-1276 and Leu-1280 in D3S6 of
Nav1.4 were important in BTX action (15, 16). For this
reason we substituted Asn-784 and Leu-788 in D2S6 and Ser-1276 and
Leu-1280 in D3S6 of Nav1.4 with Ala or Lys. The resultant
RCC values are shown in Table I. RCC values of Nav1.4-N784K
in D2S6 decreased from 0.35 to 0 compared with the wild type (Fig.
1D and Table I). Similarly, in D3S6, RCC values of
Nav1.4-S1276A and Nav1.4-S1276K also decreased
to 0.19 and 0, respectively (Table I). Mutant channels of
Nav1.4-N784A, -L788A, -L788K, -L1280A, and -L1280K did not
exhibit measurable INa. Thus, we could confirm
that Asn-784 in D2S6 and Ser-1276 in D3S6 are also critical in GTX
action, much like their role in BTX action, thus indicating that the
sites in all S6 transmembrane segments may cooperatively contribute to
GTX binding. GTX sites in Nav1.4 identified in our studies
are schematically summarized in Fig.
2B.

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Fig. 2.
Structure of GTX and GTX sites in
Nav1.4 sodium channel. A, structure of GTX I. Numerals indicate number of carbon atoms in the GTX I
molecule. B, summary of GTX sites in the Nav1.4
sodium channel. The indicated point mutations in Nav1.4
caused moderate (squares) or marked suppression
(squares with bold borders) of GTX action. For reference,
amino acid sequences (numbered from the N terminus) for the S4-S5
linkers in domains D1 and D4 and GTX sites in D1S6 and D4S6 (19-21,
24) are also displayed.
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Protocol That Derives kon and koff of GTX
Binding--
In a previous communication, we presented a
method that enables us to independently measure rate constants
kon and koff for toxin
binding and unbinding to sodium channels (10). GTX modifies sodium
channels in proportion to the time integral of the unmodified sodium
channel conductance (ToNa; Fig.
3B). The
IGTX-ToNa relationship can be readily obtained since
ToNa varies as a function of the
potential of the three conditioning pulses employed to open unmodified
sodium channels (Fig. 3A). Increment of
IGTX brought about by the three conditioning
pulses is plotted against integrated currents evoked by the three
conditioning pulses divided by the driving force (Fig. 3B).
The slope of the observed linear relationship (Fig. 3B)
gives kon according to Equation 1 (see Yuki
et al. (10) for further details),
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(Eq. 1)
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where IGTX represents current through
GTX-modified sodium channels, INa is current
through unmodified sodium channels, Etest is the
voltage applied during test pulses to evoke
IGTX, ENa is sodium
reversal potential, and C is a constant of 1.61 × 10
3 that is invariant over sodium channel isoforms
(19) and derived from sodium channel properties such as single channel
conductance and open probability of unmodified or GTX-modified sodium
channels. Briefly, numbers of GTX-modified sodium channels change
according to Equation 2 because GTX only binds to open state of the
sodium channel,
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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where O and G* represent the
number of open unmodified and GTX-modified sodium channels,
respectively, [GTX] is the concentration of GTX,
gNa and gGTX are the
single channel conductances of unmodified and GTX-modified sodium
channels, respectively, Po GTX is the open
probability of GTX-modified sodium channels, and
Econd is the voltage applied during conditioning
pulses to induce GTX-modification. Because G* and
O can be obtained from experimental data using Equations 3
and 4, Equations 2, 3, and 4 can be combined and solved for
IGTX to yield Equation 5,
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(Eq. 5)
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However, because best-fitting values of
koff were negligible compared with the term
C × kon (i.e.
koff = 0.046 and C × kon = 6.24 in the presence of 300 µM GTX (10)), we simplified Equation 5 to Equation 1 by
setting koff = 0, which thus provides an
acceptable estimate of IGTX. In fact, a linear
relationship between ToNa and
IGTX, passing through the origin, is invariably
obtained (Fig. 3B).

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Fig. 3.
Measurement of
kon and koff.
A, protocol for the measurement of
kon. An initial pulse of 40 ms (from a holding
potential of 120 mV to 80 mV) evoked modified current
(IFirst) after various prepulses induced channel
modification. Additional modification was produced by three subsequent
conditioning pulses that opened unmodified sodium channels. The
increment in modified current (IGTX) caused by
the three additional pulses (ISecond) was assessed
at 80 mV as IGTX = ISecond IFirst. Increments in modified currents caused by
trains of three conditioning prepulses to various potentials ( 70 to 0 mV in 10-mV step; see the inset in A) are shown
in B as a plot of ToNa (time integral
of unmodified currents during the three conditioning prepulses divided
by the driving force) against IGTX.
kon was measured as the slope of the fitted line
(see Equation 1 for further details). C, to measure
koff, the time constant ( ) for decay of
modified sodium currents was measured at 80 mV; for this experiment,
channel modification was induced by 100 prepulses (pulse potential, 0 mV; holding potential, 120mV). A single exponential curve was fitted
to the decay of modified sodium current. The reciprocal of this time
constant is equal to koff; i.e.
koff = 1/ (see "Results"). The
dissociation constant (Kd) is calculated from the
ratio koff:kon,
i.e. Kd = koff/kon.
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To obtain koff we measured the decay time
constant of IGTX at a potential of
80 mV (Fig.
3C). At this potential, unmodified sodium channels will not
open, and hence, no further channel modification can occur. At the same
time, because GTX-modified channels do not inactivate and in fact
maintain a constant open probability at this potential, decay of
IGTX can only represent dissociation of GTX from
its binding site. Thus, the reciprocal of this time constant can be
regarded as koff.
Measurements of kon, koff, and Kd
of GTX Binding to Wild Type Nav1.4 and Its Mutant Sodium
Channels--
Technically, it is impossible to measure
kon and koff in mutant
channels that have no sensitivity to GTX. Thus, we measured kon and koff of the
mutant channels whose sensitivity was decreased partially, and data are
summarized in Table I and Fig. 4. As an
overview, these data have four prominent features. 1) Mutations at
Phe-1579 give the largest effect on kon. 2)
Mutations at Tyr-1586 give the largest effect on
koff. 3) The change in
koff produced by mutations at Phe-1579 are
balanced by the change in kon, resulting in
relatively constant Kd. 4) Mutations at Tyr-1586
have relatively minor effects on kon, resulting
in a significant increase in Kd. These data indicate
that association and dissociation of GTX with its binding site must be
governed by different molecular entities.

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Fig. 4.
Normalized values of
kon (A),
koff (B), and
Kd (C). Values of
kon, koff, and
Kd of mutant channels are normalized to those of
wild type Nav1.4 and are displayed separately in
A, B, and C, respectively.
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Effects of Various Mutations at Phe-1579 and Tyr-1586 on
kon, koff, and Kd--
As described
above, we found that kon was increased most
markedly by mutations at Phe-1579 and koff was
increased most markedly by mutations at Tyr-1586. Thus, it is
reasonable to assume that Phe-1579 and Tyr-1586 have particularly
important roles in GTX binding. We introduced systematic mutations at
these sites using substituent amino acids with hydrophobic, aromatic,
charged, and polar side groups. Specifically, we substituted Phe-1579
with Lys, Ser, Gly, Ala, His, Cys, Met, Val, Tyr, and Trp and
substituted Tyr-1586 with Glu, Lys, Ser, Gln, Ala, Cys, Met, Ile, Phe,
and Trp. We could not detect measurable INa in
mutations F1579E, F1579Q, F1579I, F1579L, F1579P, Y1586D, Y1586R,
Y1586N, Y1586T, Y1586H, and Y1586P. Resultant RCC,
kon, koff, and
Kd values are summarized in Table
II. kon,
koff, and Kd values
normalized to those of the wild type Nav1.4 are also
graphically displayed in Fig. 4. The order of amino acids presented is
based on the degree of hydrophobicity (29-31). Only one mutant of
Y1586K in the systematic substitution experiment lost the sensitivity
to GTX, so that kon and
koff could not be estimated.
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Table II
RCC, kon, koff, and Kd in point mutants
of Nav1.4 at Phe-1579 or Tyr-1586
NA means that the GTX modification was so small that it was not
available to measure relative chord conductance,
kon, or koff.
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In mutating Phe-1579, we could not correlate the observed changes in
kon and koff with side
chain properties of amino acids such as their hydrophobic, aromatic, or
polar nature or their ionic charge. However, we did find a prominent
increase in kon especially with amino acid
substituents having small side chains (Gly, Ala, Ser, and Cys). Large
molecular dimension or ionic charge of side chains might be a hindrance
to GTX binding at Phe-1579. On the other hand, we found that
substituents with extremely hydrophilic residues (Glu, Lys, and Ser) at
Tyr-1586 significantly reduced the sensitivity to GTX. Hydrophobicity
(Y1586A, Y1586C, Y1586M, and Y1586I) may help to maintain sensitivity
to GTX, but aromatic residues (Tyr, Phe, and Trp) are most effective in
minimizing the Kd value. One of neutral residues,
Gln, at Tyr-1586 could not be a hindrance to GTX binding.
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DISCUSSION |
In this study, we utilized a new protocol that independently
determines kon and koff
values for GTX in sodium channel mutants. We, thus, uncovered new
kinetic aspects of GTX sites in Nav1.4 channels. This
protocol was remarkably effective in revealing the kinetic properties
of GTX, because it detected changes in the response of sodium channel
mutants to GTX when Kd of these mutants was not
altered compared with the wild-type control. Accordingly, we found two
functional sites for GTX action, Phe-1579 and Tyr-1586, which
specifically affect GTX binding rate constants kon and koff,
respectively. Systematic amino acid substitutions within these two
sites revealed that hydrophobic or aromatic amino acids in
Nav1.4-Tyr-1586 are required to maintain GTX binding, whereas mutations at Nav1.4-Phe-1579 do not alter the
apparent GTX binding affinity (Kd) but in fact
induce simultaneous changes in both kon and
koff. In addition to previously identified GTX
sites, we have discovered novel GTX sites in D2S6 (Asn-784) and D3S6
(Ser-1276), indicating that the GTX receptor consists of the S6
transmembrane segments of four homologous domains facing the
ion-conducting pore.
Advantages of Measuring kon and koff in the
Analysis of GTX Responses of Point Mutants--
Advantages of our
newly developed method to independently measure
kon and koff of GTX
include the following. We could detect latent mutants that affect GTX
binding. For example, many of the Phe-1579 mutants having
Kd values similar to the wild type (see Fig. 4 and
Table II) could be classified as GTX sites with functions either to
control access of toxin to the receptor site or to serve as the toxin
binding site per se. The most prominent mutation-induced
increases of kon (F1579S or F1579G) and
koff (Y1586E) occurred at distinct sites. More
precisely, systematic mutations at these two sites (Fig. 4,
A and B) revealed that mutations at
Nav1.4-Phe-1579 essentially increase
kon, and those in Nav1.4-Tyr-1586 essentially increase koff. This for the first
time gives an indication that binding and dissociation of GTX might be
regulated at two different sites within the sodium channel protein.
Assessment of Newly Derived Parameters kon and
koff--
We previously adopted the convention of RCC to
assess the sensitivity of sodium channels to GTX (19-21, 24, 28). In
the present study, we estimated Kd directly from
kon and koff as an index
of channel sensitivity to GTX. As shown in Fig. 5, a linear regression was found between
Kd and RCC, indicating that both indices reliably
report the GTX sensitivity. However, data points derived from mutants
of the 1579 residue scattered well away from the regression line (Fig.
5). This is probably due to underestimation of RCC, because unmodified
INa without preconditioning pulses in 1579 mutants was overestimated due to a residual non-inactivating current
component; such a residual component was relatively large in 1579 mutants and disappeared when GTX was depleted from internal solution
(data not shown). Without preconditioning pulses, infrequent
depolarizing pulses did not normally induce detectable GTX modification
having the described non-inactivating component, but very large
increases in kon in these mutants enabled rapid
channel modification to occur during the first pulse (see the data from
F1579A in Fig. 1, B1). Indeed, the mutants having small
molecule amino acids (Gly, Ser) at Phe-1579 exhibited measurable
non-inactivating component at the first depolarization (data not
shown). On the other hand, F1579Y was exceptional; its behavior
deviated in a direction markedly different from all other 1579 mutants.
The F1579Y mutant exhibited a peculiar phenomenon, i.e. both
peak current and GTX-modified sustained currents were spontaneously
increased during the course of experiments by a factor of 1.5 relative
to the unmodified peak current obtained at the beginning of the
experiments. By the RCC protocol, relative chord conductance of the
GTX-modified current (with prepulses) was obviously overestimated,
because unmodified INa was only sampled at the
beginning of experiments when the amplitude of unmodified
INa was 1.5 times smaller than that of the
unmodified INa that was actually present during
subsequent measurement of GTX-modified INa. We
do not know why F1579Y mutant exhibited this spontaneous increase in
INa (Fig. 5). The newly developed method, which
allows direct evaluation of kon and
koff, has the virtue of being less sensitive
than the RCC convention to perturbations such as spontaneous variations
in INa or unexpectedly rapid development of GTX
modification.

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Fig. 5.
Correlation between RCC and binding constant
(K). The parameter K is the reciprocal
of Kd. Open circles indicate values (RCC
and parameter K) obtained from mutants of position Phe-1579,
and filled squares indicate those of wild type
Nav1.4 and the indicated mutants. The regression line is
through filled squares, including wild type
Nav1.4. Note that the data points for Phe-1579 mutants
scatter away from this line.
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Specific Amino Acid Requirements in the Sites of
Nav1.4-Phe-1579 and Nav1.4-Tyr-1586 in
Achieving Interaction with GTX Molecules--
We introduced systematic
amino acid substitutions for Phe with Lys, Ser, Gly, Ala, His, Cys,
Met, Val, Tyr, and Trp in Nav1.4-Phe-1579 and for Tyr with
Glu, Lys, Ser, Gln, Ala, Cys, Met, Ile, Phe, and Trp in
Nav1.4-Tyr-1586 consisting of hydrophilic, hydrophobic, aromatic, and polar amino acid side chains.
At the Nav1.4-Phe-1579 site there was no clear indication
that hydrophobic, polar, or aromatic properties of residue side chains
play a critical role in increasing either kon or
koff. It seems that larger molecular size of the
amino acid side chains predicted smaller rate constants. Thus, the
emerging picture is that the smaller the molecular size of the side
chain, the greater the access of GTX molecules to the receptor site. As
for the site at Tyr-1586, it is distinct from the site at Phe-1579 in
that the GTX sensitivity (Kd = koff/kon) was extensively
altered by the mutations. Furthermore, we could categorize the effect of mutations into three groups according to the resultant
Kd values. Aromatic residues (Tyr, Phe, Trp) produce
the highest sensitivity of the three groups, although an exception was
found in the case of Gln. Hydrophilic residues (Glu, Lys, Ser) could not maintain a suitable microenvironment to permit GTX binding. Hydrophobic residues (Ala, Cys, Met, Ile) improved the efficiency of
GTX binding compared with hydrophilic residues.
It has been reported that specific amino acids are required in BTX or
local anesthetic binding to a putative receptor site in D4S6
(Nav1.3-F1710) corresponding to Nav1.4-Phe-1579
(9, 32). Stable BTX binding to the channel protein requires a polar or
aromatic residue at this receptor site to maintain the ion-dipole or
cation-
interaction (9). Tetracaine requires an aromatic residue at
its receptor site to maintain electrostatic interactions between the
positive charge on the protonated tertiary amine of this local
anesthetic and the electron-rich
face of the aromatic residue (32).
As for GTX, Yakehiro et al. (8) show that the hydrophobic
moiety must be preserved on the
-surface of GTX molecules for
effective GTX action because the introduction of a hydroxyl group into
C-14S facing the
-surface of the GTX analog markedly reduced GTX potency (8). Thus, it is supposed that the hydrophobic environment around the receptor site is important in GTX action. In
this study, we argued that an aromatic or hydrophobic side chain at
residue Tyr-1586 in Nav1.4 is required to preserve
sensitivity to GTX. This is consistent with the requirement for
hydrophobicity on the
-surface of GTX in maintaining GTX potency.
Direct interaction between the
-surface of GTX and Tyr-1586 may be
necessary for normal GTX action.
Although Tyr-1586 has a determinant role in GTX action as discussed
above, Phe-1579 may have a rather regulatory function. Mutations at
Nav1.4-Phe-1579 produced only a marginal change in Kd values. Furthermore, F1579K was the only mutant
whose sensitivity was not totally eliminated by Lys substitution (see Table I). It is reasonable to assume that the charged side chain in
F1579K interferes with access of the hydrophobic
-surface of GTX
molecules because we observed an exceptionally small
kon when Lys was substituted (see Fig.
4A).
Properties That Differentiate GTX from Other Site 2 Toxins or Local
Anesthetics--
GTX shares common binding sites with the Site 2 toxins BTX and veratridine, namely, Ile-433, Asn-434, and Leu-437 in
D1S6 and Ile-1575 and Phe-1579 in D4S6 of Nav1.4 (17-23).
Other GTX-specific sites were further documented in D4S6 (Tyr-1586) of
Nav1.4 (20). Several GTX sites were also found in D1 and D4
(see Fig. 2B) (19-21, 24). This study has now added two
novel sites in D2S6 (Asn-784) and D3S6 (Ser-1276) to the list of
binding sites common to Site 2 toxins. However, Tyr-1586 is
GTX-specific, because mutations at this position did not affect BTX
binding (17, 22). This might explain the difference in binding kinetics
between GTX and BTX. BTX dissociates from the open state of the sodium
channel (9), whereas GTX exclusively dissociates from the closed state (10). It is supposed that BTX dissociates from its receptor site in the
ion-conducting pore and shifts to a cytoplasmic membrane site during
the open state of the channel, as if it were reversing its itinerary
(9). On the other hand, because Tyr-1586 is GTX-specific among Site 2 toxins and mutation of this site dramatically increases unbinding
(koff) of GTX, it is possible that this site
contributes to the GTX-specific binding-unbinding reaction by trapping
GTX in the open channel and releasing GTX from the closed channel.
Local anesthetics like lidocaine, tetracaine, QX314, and QX222 modify
channel activation and block sodium current. These local anesthetics
also share common binding sites at Asn-434 and Leu-437 in D1S6,
Ser-1276 in D3S6, Ile-1575, Phe-1579, and Tyr-1586 in D4S6 of
Nav1.4 with GTX (16, 19-21, 33, 34). As in GTX binding, local anesthetic binding is affected by introduction of
amino acid substitutions at both Phe-1579 and Tyr-1586. However, there are also differences in mode of reactions between GTX and local anesthetics. In contrast to GTX reactions, Phe-1579 is thought to be
the major local anesthetic binding site rather than Tyr-1586, which
merely regulates access of local anesthetics to the Phe-1579 site
(32).
Other Critical Sites for GTX Action in S6 Transmembrane
Segments--
Much as we determined that sites Phe-1579 and Tyr-1586
in D4S6 are critical to GTX action on the basis of drastic changes in
kon or koff, we are
likely to find similarly important sites in other domains. However, in
this study, mutants in other domains did not produce a prominent change
in either kon or koff.
There are two reasons for failure to detect these sites; 1) we cannot determine kon or koff in
mutants that are completely insensitive to GTX, thus exhibiting RCC
values of 0, as observed especially with Lys mutations at Ile-433,
Asn-434, Leu-437, Asn-784, and Ser-1276 and 2) mutations such as I433V
do not necessarily change the chemical properties of relevant amino
acids (i.e. both Ile and Val are hydrophobic residues).
Thus, application of systematic mutation to these unexplored sites with
measurement of kon and koff might be a productive avenue for future
research. Ile-433 of D1S6 could be one of the most promising sites,
because mutations of this site affect binding not only of GTX and other
Site 2 toxins but also of pyrethroids (35). It is interesting to note
the common functional effects of GTX and pyrethroids on sodium channel kinetics; both GTX and pyrethroids markedly prolong channel open time and remove or slow sodium inactivation (36-38).
 |
ACKNOWLEDGEMENTS |
We express our sincere gratitude to Dr.
Stephen M. Vogel (Department of Pharmacology, University of Illinois at
Chicago, College of Medicine) for critical reading of the
manuscript. We thank the Research Center for Molecular Medicine,
Faculty of Medicine, Hiroshima University for the use of their facilities.
 |
FOOTNOTES |
*
This work was supported by Ministry of Education and Culture
of Japan Grants 11470011 and 14370013 (to K. Y.) and 11770023 and
14770014 (to E. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-82-257-5123; Fax: 81-82-257-5124; E-mail:
kyamaok@hiroshima-u.ac.jp.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M212133200
2
Each mutant channel will be referenced by the
original amino acid followed by its number and introduced amino acid.
 |
ABBREVIATIONS |
The abbreviations used are:
BTX, batrachotoxin;
D, domain;
GTX, grayanotoxin;
INa, Na+ current;
I-V, current-voltage;
S, segment;
RCC, relative chord conductance.
 |
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