From the Institute of Molecular Cardiobiology, The
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the ** Department of Microbiology and Molecular Genetics,
University of California, Irvine, California 92697
Received for publication, October 23, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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µ-Conotoxins (µ-CTXs) block skeletal
muscle Na+ channels with an affinity 1-2 orders of
magnitude higher than cardiac and brain Na+ channels.
Although a number of conserved pore residues are recognized as critical
determinants of µ-CTX block, the molecular basis of isoform-specific
toxin sensitivity remains unresolved. Sequence comparison of the domain
II (DII) S5-S6 loops of rat skeletal muscle (µ1,
Nav1.4), human heart (hh1, Nav1.5), and rat
brain (rb1, Nav1.1) Na+ channels reveals
substantial divergence in their N-terminal S5-P linkers even though the
P-S6 and C-terminal P segments are almost identical. We used
Nav1.4 as the backbone and systematically converted these
DII S5-P isoform variants to the corresponding residues in
Nav1.1 and Nav1.5. The
Nav1.4 µ-Conotoxins (µ-CTXs)1 are guanidinium toxins that
are produced by the sea snail Conus
geographus (1-5) and block Na+ channels by physical
occlusion of the pore (2, 6-10). Although chemically dissimilar to the
pufferfish-derived tetrodotoxin (TTX) and the red tide saxitoxin (STX),
µ-CTXs show biological actions similar to those of TTX and STX. Like
TTX and STX, µ-CTXs block Na+ channels from different
tissues with vastly different affinities. µ-CTXs preferentially block
skeletal muscle and eel Na+ channels with affinities 2 orders of magnitude higher than for block of the cardiac and brain
counterparts. In contrast, brain and skeletal muscle channels are 3 orders of magnitude more sensitive to TTX/STX than are heart channels
(11-15). Although the key determinant for isoform-specific TTX and STX
block has been identified (tyrosine and phenylalanine in the domain I
(DI) P loop of the TTX/STX-sensitive skeletal muscle and brain
isoforms, respectively, but cysteine at the homologous position in the
toxin-resistant cardiac subtype (16-18)), previous attempts to study
isoform-specific µ-CTX block have been largely unsuccessful because
its determinants are more widespread and have a much larger footprint
in the channel pore than the compact TTX and STX (8-10, 14, 15,
19-23).
The S5-S6 linkers that form the outer pore vestibule and the
selectivity filter are divided into three regions: S5-P, P loop (whose
descending and ascending regions are referred to as SS1 and SS2,
respectively), and P-S6 (see Fig. 1A) (11). Even though homologous regions are found in all four S5-P linkers of different Na+ channel isoforms, those of domains I-III display
substantial divergence. DIII and DIV have small variable regions distal
to the highly conserved P loops. To date, all critical determinants for
µ-CTX block identified in the conventional aqueous pore formed by the
P loop SS2s (11) and the P-S6 linkers (10, 24) are residues that are
invariant in cardiac, skeletal muscle, and brain channels (8-10, 23).
Therefore, they cannot explain the vast differences in isoform-specific
µ-CTX sensitivity. Chimeric studies have demonstrated that DI and
especially DII are more critical determinants of isoform-specific
µ-CTX affinity than are DIII and DIV (the rat skeletal muscle
(Nav1.4) Na+ channels carrying the entire DI or
DII from the human heart (Nav1.5) isoform were ~20- and
>200-fold less sensitive, respectively, than wild-type (WT)
Nav1.4) (14, 15); however, the detailed molecular basis
remains unresolved. Sequence inspection of the DII S5-S6 loops of the
toxin-sensitive Nav1.4 and the toxin-resistant rat brain
(Nav1.1) and Nav1.5 Na+ channels
reveals substantial divergence in their S5-P linkers, although the
C-terminal portions (i.e. P and P-S6) that form the conventional ion-conducting pore are almost identical (for sequence comparisons, see Figs. 1B and 4A). Because many
of the known toxin determinants are clustered in the DII pore region
(8-10, 23), the divergence of the adjacent DII S5-P linker motivated
us to test whether the differences therein might underlie
isoform-specific µ-CTX block. To test this hypothesis, we
systematically converted the isoform-specific variants in
Nav1.4 to those in Nav1.1 and Nav1.5 and vice versa and then assayed their effects on
µ-CTX sensitivity. We identified DII S5-P variants that prominently determine isoform-specific µ-CTX block. The molecular basis of isoform-specific µ-CTX block is discussed in the context of
structural implications of the sodium channel pore. A preliminary
report has appeared (25).
Site-directed Mutagenesis and Heterologous Expression--
The
gene encoding Nav1.4 or Nav1.5 sodium channels
was cloned into the pGFP-IRES vector with an internal ribosomal entry
site separating it from the green fluorescent protein (GFP) reporter gene (26); that of Nav1.1 was cloned into pLCT1 (27).
Mutagenesis was performed using PCR with overlapping mutagenic primers.
The presence of the desired mutation(s) was confirmed by DNA
sequencing. Nav1.4 and Nav1.5 channels were
transfected into tsA-201 cells using LipofectAMINE Plus (Invitrogen)
according to the manufacturer's protocol. Briefly, plasmid DNA
encoding the WT or mutant Electrophysiology and Data Analysis--
Electrophysiological
recordings were performed at room temperature using the whole cell
patch clamp technique (29) for tsA-201 cells and two-electrode voltage
clamp recordings for oocytes. Transfected tsA-201cells were identified
by epifluorescence microscopy from the coexpressed green fluorescent
protein. Patch pipette electrodes had final tip resistances of 1-3
megohms; the internal recording solution contained (in mM):
35 NaCl, 105 CsF, 1 MgCl2, 10 HEPES, 1 EGTA, pH adjusted to
7.2 with CsOH. For oocyte recordings, the pipette contained 3 M KCl and had final tip resistances of 2-4 megohms. All
electrical recordings were performed in a bath solution containing (in
mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH.
Designated amounts of µ-CTX were added to the bath when required.
Toxin was superfused continuously during the experiment at ~10 ml/min
(bath volume was 150 µl). Raw current records were analyzed using
custom written software. Half-blocking concentrations
(IC50) for µ-CTX were determined by fitting the
dose-response data to the binding isotherm,
All data reported are the mean ± S.E. Statistical significance
was determined using a paired Student's t test at the 5% level.
Amino Acid Differences in the Conventional Pore Are Not Responsible
for Isoform-specific µ-CTX GIIIB Sensitivity--
We first compared
the µ-CTX GIIIB sensitivity of WT Nav1.4 and
Nav1.5 Na+ channels (Fig.
1B). Consistent with previous
studies (12, 14, 15, 30), Nav1.4 (IC50 = 29.7 ± 8.6 nM; n = 7) was
substantially more susceptible to µ-CTX block than Nav1.5
(IC50 = 15.3 ± 7.7 µM;
n = 3; p < 0.01). This vast
isoform-specific difference in toxin block prompted us to investigate
the underlying molecular basis. Even though the P loops of
Nav1.4 and Nav1.5 Na+ channels are
highly conserved, a number of differences do exist. One such difference
is the residue at position 401 in DI (Nav1.4 numbering
throughout this manuscript unless otherwise specified). Although
replacing Tyr401 with a cysteine could largely explain the
isoform-specific differences in TTX and STX block observed with
Nav1.4 and Nav1.5 channels (16, 18), this
mutation had little effect on µ-CTX block (9, 14). Despite the lack
of a significant role for this pore residue, it is still conceivable
that other differences in the pore, especially those involving a charge
difference, may be responsible for isoform-specific µ-CTX block.
Charge-variant residues in the pore include DIII Glu1251,
DIII Lys1252, and DIV Asp1545 (Gly, Tyr, and
Tyr, respectively, at the equivalent locations in Nav1.5).
To examine the role of these residues in the isoform specificity of
µ-CTX, we created and studied the Nav1.4 constructs E1521G/K1252Y and D1545Y. Both E1521G/K1252Y (IC50 = 53.6 ± 17.6 nM, n = 3) and D1545Y
(IC50 = 54.7 ± 9.1 nM, n = 3) channels displayed Nav1.4-like sensitivity to µ-CTX
GIIIB (p > 0.05), indicating that these variants in
the conventional pore are not responsible for isoform-specific µ-CTX
block of Nav1.4 and Nav1.5 Na+
channels.
DII S5-P Linker Plays a Role in Isoform-specific µ-CTX Block of
Nav1.4 and Nav1.5 Na+
Channels--
The lack of effect observed with isoform-specific
mutations in the conventional pore prompted us to examine other channel regions. Comparison of the primary sequences of Nav1.4 and
Nav1.5 reveals substantial divergence in the DII S5-P
linkers. Sequence alignment of the two isoforms, however, is ambiguous
because the Nav1.5 linker is shorter than that in
Nav1.4, and the remaining residues contain numerous
conservative differences. Fig. 1B shows the two alignments
that we used as a guide to create DII S5-P Nav1.4
constructs (isoform-specific residues mutated are identified by
asterisks). Fig. 1C summarizes the effects of
these variant mutations on µ-CTX GIIIB block. All DII S5-P mutants
except A DII S5-P Nav1.5 Chimeric Construct Displayed Enhanced
Sensitivity to µ-CTX--
If the DII S5-P isoform-specific variants
identified above are truly responsible for isoform-specific µ-CTX
block of Nav1.4, then conversion of the equivalent residues
in Nav1.5 to those in Nav1.4 should enhance the
sensitivity of the toxin-resistant heart channels. However, it should
be pointed out that no single residue completely reproduced the cardiac
sensitivity. The closest was D730S, whose "isoform specificity" is
somewhat debatable because the sequence alignment in this region is
ambiguous, as shown in Fig. 1B, and because of the seemingly
important steric role of DII S5-P, as gauged by the mutations at
position 728. We postulate that the spacing between these residues
plays a role in high affinity toxin block of WT Nav1.4.
Therefore, we created a Nav1.5 Different DII S5-P Variants Underlie Isoform-specific µ-CTX Block
of Nav1.1 and Nav1.4--
Although the amino
acid differences between Nav1.4 and Nav1.5 at
positions 724, 725, 728, 730, and 731 in the DII S5-P linker appear to
modulate µ-CTX block prominently, these residues are absolutely
conserved between Nav1.1 and Nav1.4 (Fig.
4A) and thus cannot explain
their known difference in µ-CTX sensitivity (11, 12). Further
sequence comparison of the DII S5-P linker of the muscle and brain
isoforms reveals two other unique variants at positions 729 and 732:
serine and asparagine in Nav1.4 compared with threonine and
lysine in Nav1.1. To test their roles, we replaced the
Nav1.4 residues Ser729 and Asn732
with those in Nav1.1 and studied their effects (Fig. 4,
B and C). Consistent with the notion that the DII
S5-P linker plays a prominent role in determining the isoform-specific
µ-CTX response, the chimeric single mutations S729T (IC50 = 100.1 ± 22.4 nM, n = 3) and N732K
(IC50 = 249.6 ± 100.7 nM,
n = 8) rendered Nav1.4 more
"brain-like" by reducing their sensitivity. Similar to N732K, replacement of residue 732 with an arginine (N732R), which also places
a positive charge at the same position but with a slightly larger side
chain volume (148 Å3 versus 135 Å3
of lysine), also reduced GIIIB block (IC50 = 151.1 ± 25.8 nM, n = 3; Fig.
5). Combining S729T and N732K mutations
(S729T/N732K) further decreased µ-CTX sensitivity (IC50 = 904.9 ± 286.0 nM, n = 4) to a level
comparable with that of WT Nav1.1 (IC50 = 2686.5 ± 138.8 nM, n = 6; Fig. 4,
B and C), suggesting that the effects of these
mutations were additive.
To confirm the isoform-specific role of residues 729 and 732, we next
converted the analogous residues in Nav1.1 simultaneously to those of Nav1.4 (i.e.
Nav1.1-T925S/K928N). Indeed, this chimeric double mutation
reproduced the high affinity µ-CTX GIIIB block observed with
Nav1.4 channels (IC50 = 52.8 ± 10.6 nM, n = 4; Fig. 4, B and
C). Collectively, our data substantively bolster the notion
that the DII S5-P linker is a determinant for isoform-specific µ-CTX block.
Differential Sensitivities to µ-CTX GIIIA and GIIIB--
Even
though µ-CTX GIIIA and GIIIB have nearly identical backbone
structures and block WT Nav1.4 with the same affinity, we recently reported that the negatively charged DII P-S6 residues Asp762 and Glu765, when mutated to lysine,
confer upon Nav1.4 channels the unique ability to
distinguish between these toxin forms (20, 31-33). Because the DII
S5-P variants identified are also in DII (in particular, the Asp Effects of N732K/D762K/E765K on µ-CTX GIIIA
and GIIIB Sensitivities--
Reversing both the DII P-S6 negative
charges at positions 762 and 765 by lysine substitution (D762K/E765K)
results in an additional decrease in GIIIB (but not GIIIA) block
compared with the individual single mutations alone, suggesting that
both charges are required simultaneously for high affinity GIIIB block
(10, 20). We next investigated whether a similar additive effect was
observed when N732K from DII S5-P and the DII P-S6 mutations are
combined. The triple mutation N732K/D762K/E765K rendered
Nav1.4 channels highly insensitive to µ-CTX GIIIB
(IC50 = 110 ± 25 µM; n = 4) but only modestly reduced GIIIA block (IC50 = 182.4 ± 90.8 nM; n = 3). Fig.
6 shows a typical toxin experiment
performed with this channel construct. Application of 5 µM GIIIB to N732K/D762K/E765K channels produced no
significant current blockade, and 10 µM GIIIB blocked
INa only to 92.4 ± 4.5% of the control
toxin-free level (versus ~87% block of D762K/E765K with 3 µM GIIIB; see Fig. 4 of Ref. 20). Thus, the effect of
N732K on GIIIB block and those of the DII P-S6 mutations were largely
additive. In contrast, application of 300 nM GIIIA to
N732K/D762K/E765K mutant channels substantially reduced
INa to 33.8 ± 10.6%. Therefore,
N732K/D762K/E765K, as anticipated from the blocking phenotypes of the
individual mutations, could also discriminate between GIIIA and
GIIIB.
Ion channels with similar ionic selectivity may differ
significantly in other properties related to the pore, such as drug and
toxin sensitivity. As mentioned, skeletal muscle Na+
channels exhibit sensitivity to block by µ-CTX which is 2 orders of
magnitude greater than their cardiac and brain counterparts. Although
previous studies have identified critical pore residues for high
affinity µ-CTX block (8-10, 22-24, 34), these residues are well
conserved among different channel isoforms and therefore cannot explain
their vastly different toxin sensitivities. To look for the molecular
components responsible for such isoform-specific differences, we
switched our focus to the DII S5-P linker, a highly variable region
lying N-terminal to the highly conserved conventional pore formed by
the P loop and the P-S6 linker (11, 24). Although previous studies of
Na+ channels have mostly focused on the conserved pore
region, the S5-P linkers of other ion channels have been reported to
regulate properties such as toxin sensitivity, gating, and permeation
(28, 35, 36). In this study, we provided evidence that isoform-specific variants in the DII S5-P linker of Na+ channels play a
prominent role in shaping their distinctive isoform-specific sensitivity to µ-CTX block. Although not explored here, changes of
gating properties (particularly of current decay) were also observed
with some of these DII S5-P variant mutants (e.g. A728S and
H-to-M chimera). Taken collectively, our present results further highlight the functional importance of this extracellular linker.
Different Molecular Bases Underlie Isoform-specific µ-CTX Block
of Nav1.4, Nav1.5, and Nav1.1
Na+ Channels--
Interestingly, all of the sequence
variants that alter µ-CTX block of Nav1.4 and
Nav1.5 (i.e. Val724,
Cys725, Ala728, Asp730,
Cys731) are absolutely conserved between Nav1.1
and Nav1.4 channels. However, we found that the variants
between Nav1.1 and Nav1.4 within the same
linker at positions 729 and 732 also modulate µ-CTX block. These
results are consistent with previous chimeric studies demonstrating
that DII plays the most important role in determining isoform-specific
µ-CTX block (15). Taken collectively, it is clear that amino acid
differences in the DII S5-P linkers of Nav1.1,
Nav1.4, and Nav1.5 channels figure prominently
in their distinctive isoform-specific µ-CTX sensitivities. This
notion is bolstered by the enhanced µ-CTX sensitivity observed with
the toxin-resistant Nav1.1 and Nav1.5 channels
when the converse DII S5-P chimeric mutations were introduced. However,
although the B-to-M Nav1.1 double mutant reproduced the
high affinity blocking phenotype of Nav1.4, the H-to-M
Nav1.5 chimeric construct only modestly enhanced µ-CTX
block of the cardiac channel. These observations suggest that the basis
for isoform-specific µ-CTX block is more complex in the cardiac
channel and is likely to involve as yet other unidentified structural
determinants. Other channel regions (e.g. DI (15) and its
S5-P linker in particular) are likely to be involved in modulating the
isoform specificity of Nav1.5. On the other hand, the weak
gain-of-function phenotype may simply reflect the exacting nature of
such experiments, in which one of many subtle structural features may
suffice to undermine high affinity block. Nonetheless, it is obvious
that the toxicological profiles of Nav1.1,
Nav1.4, and Nav1.5 Na+ channels
have different molecular bases, but all appear to involve the same
channel region (i.e. the DII S5-P linker).
Discrimination between µ-CTX GIIIA and GIIIB--
The isoform
variant N732K is unique compared with other DII S5-P determinants
because it enabled Nav1.4 channels to distinguish between
GIIIA and GIIIB (see Ref. 37). The importance of residue 732 in
determining the ability of Na+ channels to discriminate
between the two toxin isoforms is illustrated further by our finding
that WT Nav1.1 but not Nav1.1 T925S/K928N channels were blocked differentially by GIIIA and GIIIB. Collectively, our present results further exemplify the principle of "latent specificity," in which the ability of a receptor to recognize homologous ligands can be rendered highly specific (or abolished) by
engineering the amino acid backbone even in the absence of innate
specificity (20). In fact, the ~170-fold difference in GIIIA and
GIIIB sensitivities observed with the triple mutant N732K/D762K/E765K
was by far the largest among those that we have reported
(i.e. D762K, E765K, D762K/E765K, D762Q/E765Q) to possess this unique discriminative ability (20). Our results may have pragmatic
implications for future biosensor design.
We have shown previously that combining the individual DII P-S6
charge-reversed mutations D762K and E765K (i.e. D762K/E765K) further destabilizes GIIIB block compared with the corresponding single
mutations alone. However, the additional reduction is only ~4-fold, a
relatively small change compared with the ~200-fold decrease observed
with the individual single substitutions (10, 20). These observations
highlight the plausibility that the DII P-S6 residues may have common
and/or preferential interacting toxin partners (19, 20). In contrast,
we found that the ~5-fold reduction in GIIIB block observed with the
triple mutant N732K/D762K/E765K relative to D762K/E765K channels was
highly comparable with the 10-fold decrease in toxin sensitivity caused
by the single mutation N732K alone compared with WT Nav1.4,
suggesting that the effects of N732K and the DII P-S6 mutations on
GIIIB block were largely independent and additive. Cummins et
al. (37) suggest that the DII S5-S6 loop may have a
topology that allows residues 732, 762, and 765 to align along the same
axis and interact simultaneously with Arg14 of GIIIB, the
charge-change toxin variant that underlies the differential binding of
GIIIA and GIIIB to the DII P-S6 mutants (20, 37). Our data further
suggest that Lys732 is likely to influence toxin binding
via mechanisms independent of the DII P-S6 residues. Given the
improvement and reduction of block observed with N732D and N732K (and
N732R) channels, respectively, it is possible that residue 732 interacts directly with the toxin molecule and/or with other native
charged side chains of the channel. Further experiments using toxin
derivatives and mutant cycle analysis may provide additional insights
and are currently under way. Regardless of the underlying mechanism, it
is clear that the variant at position 732 plays a critical role in
determining the isoform-specific differences in µ-CTX binding between
Nav1.1 and Nav1.4 but not Nav1.5
channels and the ability of channels to distinguish between the GIIIA
and GIIIB forms of µ-CTX.
Structural and Pharmacological Implications--
Recently, French
and colleagues (38) proposed that µ-CTX inhibits Na+
currents in at least two ways. First, the physical bulk of µ-CTX (molecular weight ~2,600) sterically blocks the channel vestibule. Second, basic residues protruding from different faces of the toxin
molecule reduce the effective capture volume from which ions enter the
pore. Most importantly, the charge of Arg13 appears to
neutralize the effects of native acidic pore residues (such as
Glu758) that serve to increase the effective
Na+ concentration at the external pore mouth. Clearly, high
affinity µ-CTX binding to the pore requires optimal positioning of
residues that contribute to the toxin receptor.
The S5-P linker, N-terminal to the "descending" limb of the P loop
(SS1), is not thought to line the pore or to interact with the toxin
directly (9, 14). This notion is supported by several lines of
evidence. Asp730 is unexposed when probed by cysteine
substitution and sulfhydryl-modifying agents (14). Furthermore, there
is no observable molecular interaction (Nav1.5 variant substitutions V724R,
C725S, A728S, D730S, and C731S (Nav1.4 numbering) reduced block of Nav1.4 by 4-, 86-, 12-, 185-, and 55-fold
respectively, rendering the skeletal muscle isoform more
"cardiac-like." Conversely, an Nav1.5
Nav1.4 chimeric construct in which the Nav1.4
DII S5-P linker replaces the analogous segment in Nav1.5
showed enhanced µ-CTX block. However, these variant determinants are
conserved between Nav1.1 and Nav1.4 and thus
cannot explain their different sensitivities to µ-CTX. Comparison of
their sequences reveals two variants at Nav1.4 positions
729 and 732: Ser and Asn in Nav1.4 compared with Thr and
Lys in Nav1.1, respectively. The double mutation
S729T/N732K rendered Nav1.4 more "brain-like"
(30-fold
in block), and the converse mutation T925S/K928N in
Nav1.1 reproduced the high affinity blocking phenotype of
Nav1.4. We conclude that the DII S5-P linker, although
lying outside the conventional ion-conducting pore, plays a prominent
role in µ-CTX binding, thus shaping isoform-specific toxin sensitivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (1 µg/60-mm dish) was added to
the cells with LipofectAMINE, followed by incubation at 37 °C in a
humidified atmosphere of 95% O2, 5% CO2 for
48-72 h before electrical recordings. For Nav1.1 channels, cRNA was transcribed from NotI-linearized DNA using T7 RNA
polymerase (Promega, Madison, WI) and coinjected with the rat brain
1 subunit into Xenopus oocytes for heterologous
expression as described (9, 28).
where IC50 is the half-blocking concentration,
[toxin] is the toxin concentration, and
I0 and I are the peak currents
measured from a step depolarization to
(Eq. 1)
10 mV from a holding potential of
100 mV before and after application of the blocker, respectively. Concentrations of 30 and/or 100 nM µ-CTX were initially
used to screen mutants for changes in sensitivity. Depending on the
sensitivity, concentrations used for subsequent experiments were chosen
to bracket the IC50 of a particular mutant (e.g.
1 and 3 µM for C731S).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
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Fig. 1.
Effects of DII S5-P isoform variants
between Nav1.4 and Nav1.5 on
µ-CTX block. A, schematic diagram showing
the S5 and S6 transmembrane segments and the reentrant S5-S6 linker.
The S5-S6 linker consists of the S5-P, P loop (i.e. SS1 and
SS2), and P-S6 linker. B, comparison of the primary
sequences of the DII S5-P linker of human cardiac (Nav1.5)
and rat skeletal muscle (Nav1.4, bold)
Na+ channels. The S5-P linker is N-terminal to the
conventional aqueous pore formed by the P loop and the P-S6 linker and
displays substantial divergence among different isoforms of
Na+ channels. Sequence alignment of the two isoforms is
ambiguous because the DII S5-P linkers are different lengths. Thus,
there are many different possible alignments of this region of the
channels, two of which are shown. Asterisks indicate
isoform-specific amino acid variants within a given alignment. The
mutated Nav1.4 residues are boxed. Those that
reduced toxin sensitivity are shown in italics.
C, bar graphs summarizing the half-blocking
concentrations (IC50) of WT and DII S5-P mutant
Nav1.4 channels for block by µ-CTX GIIIB. The isoform
variants C723S, V724R, A728S, D730S, and C731S significantly reduced
block by µ-CTX. Data shown are the mean ± S.E.
Numbers in parentheses represent the number of
determinations for each of the individual bars, with
asterisks indicating statistical differences
(p < 0.05). n.e., not expressed.
723C/V724R/
725C/K726R expressed functional channels.
Because the deletion of Cys723 and Cys725 did
not yield measurable currents, we studied the roles of these residues
by serine substitution (i.e. C723S and C725S, respectively) as inspired by the Cys
Ser difference at position 731. Indeed, the
isoform-specific point mutations V724R (IC50 = 111.4 ± 14.4 nM, n = 5), C725S (IC50 = 2.5 ± 0.1 µM, n = 6), A728S
(IC50 = 364.3 ± 24.3 nM,
n = 4), D730S (IC50 = 5.5 ± 0.5 µM, n = 3), and C731S (IC50 = 1.6 ± 0.5 µM, n = 3) significantly
reduced GIIIB block of µ1 by 4-, 86-, 12-, 185-, and 55-fold
respectively, rendering the muscle isoform more "cardiac-like."
Interestingly, N732D slightly improved toxin binding. To obtain further
mechanistic insights, we also studied the mutations A728V and A728L
(Fig. 2A). Of the mutations at
the 728 position, A728L channels displayed the lowest sensitivity to
GIIIB (IC50 = 1.4 ± 0.2 µM,
n = 3) followed by A728V (IC50 = 1.1 ± 0.4 µM, n = 4), A728S, and WT
Nav1.4. Because leucine has the largest side chain (124 Å3), followed by valine (105 Å3), serine (73 Å3), and alanine (67Å3), these observations
are consistent with the notion that side chain volume at position 728 influences toxin block and that µ-CTX binding is optimal when an
alanine is present. For channel position 730, we neutralized the native
cationic residue also by replacing it with an asparagine
(i.e. D730N). Similar to D730S, D730N showed significantly
reduced GIIIB block (IC50 = 3.0 ± 0.3 µM, n = 4, p < 0.05).
Interestingly, although the charge-conserved mutation D730E
(IC50 = 553.4 ± 77.9 nM,
n = 4) improved toxin block compared with D730S and
D730N channels, the charge-reversed mutation D730K (IC50 = 1.6 ± 0.2 µM, n = 3) did not worsen
it compared with the charge-neutralized substitutions D730S and D730N,
as might have been anticipated from a simple electrostatic effect.
Instead, D730K channels were >2-fold more susceptible than either of
the neutral counterparts. These results of Asp730 mutations
are summarized in Fig. 2B.
View larger version (27K):
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Fig. 2.
Effects of multiple substitutions at
positions 728 and 730 on µ-CTX sensitivity.
A: top, representative current tracings through
WT, A728S, A728V, and A728L Nav1.4 channels with or without
µ-CTX as indicated. Middle, amino acid substitutions into
position 728. Side chains are shown without their backbone atoms in the
CPK format. Hydrogen, white; oxygen, red; carbon,
gray; nitrogen, blue. Bottom,
bar graphs summarizing the IC50 values of the
same channels for block by µ-CTX. A728V channels were less sensitive
than A728S but more sensitive than A728L. Data shown are the mean ± S.E. B: top, typical raw current traces of WT,
D730S, D730N, D730E, and D730K Nav1.4 channels with or
without µ-CTX as indicated. Middle, amino acid side chains
substituted into position 730. Bottom, IC50
summary.
Nav1.4 (heart-to-muscle or H-to-M) chimera in which 10 consecutive DII S5-P
residues from Nav1.4 were spliced into the "homologous"
region of Nav1.5 (Fig.
3A). Although falling short of
reproducing the high affinity µ-CTX block seen in Nav1.4
(Nav1.4 H-to-M IC50 = 4.2 ± 1.3 µM, n = 7), block by 5 µM
µ-CTX GIIIB was enhanced significantly in the DII S5-P
Nav1.5 chimeric construct (60.7 ± 7.5%,
n = 7) compared with the WT Nav1.5
(31.2 ± 8.9%, n = 3; p < 0.05;
Fig. 3B).
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Fig. 3.
A µ-CTX-sensitive
Nav1.5 chimeric construct. A, the
Nav1.4 DII S5-P segment 723-729/731-732 (bold)
was substituted into the Nav1.5 Na+ channels to
create the Nav1.5 H M chimera. B, comparison
of the time course of block development of WT Nav1.5 and
the DII S5-P Nav1.5 H-to-M chimeric channels by 5 µM µ-CTX. The inset shows representative raw
current traces of WT Nav1.5 and the chimeric channels
recorded with or without 5 µM µ-CTX.
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Fig. 4.
Effects of DII S5-P isoform variants between
Nav1.1 and Nav1.4 on
µ-CTX block. A, comparison of the DII
S5-P linkers of Nav1.1 and Nav1.4
Na+ channels. The critical DII S5-P variants identified for
Nav1.4 and Nav1.5 in Fig. 2 are absolutely
conserved between Nav1.1 and Nav1.4. Sequence
comparison of these isoforms reveals two other differences at
Nav1.4 positions 729 and 732. B, representative
Na+ currents through WT, S729T, N732K, and S729T/N732K
Nav1.4 channels and those through WT and T925S/K928N
(TS/KN) Nav1.1 channels recorded in the absence
and presence of µ-CTX GIIIB. C, bar graph
summarizing the IC50 for µ-CTX GIIIB block of the same
channels from B. The mutations S729T, N732K, and S729T/N732K
rendered Nav1.4 less sensitive to µ-CTX block, whereas
the converse chimeric mutation T925S/K928N rendered Nav1.1
more sensitive with an affinity similar to Nav1.4. *, p < 0.05.
View larger version (24K):
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Fig. 5.
Differential sensitivities of Na+
channels to µ-CTX GIIIA and GIIIB.
Representative Na+ current tracings through N732K and WT
Nav1.1 channels measured in the absence and presence of
GIIIA and GIIIB are shown as indicated (top panel).
Bar graphs summarizing the IC50 values of
various Nav1.4 and Nav1.1 channels for block by
µ-CTX GIIIA and GIIIB are shown in the bottom panel.
N732K, N732R, and WT Nav1.1 channels displayed differential
sensitivities to GIIIA and GIIIB (*, p < 0.05). All
other DII S5-P Nav1.4 variants as well as
Nav1.1-T925S/K928N (TS/KN) were equally
insensitive to both GIIIA and GIIIB forms of µ-CTX.
Lys N732K variant is also a lysine substitution), we tested whether our
DII S5-P variant channels possessed such discriminative ability so as
to obtain insights into this obscure channel region. Fig. 5 shows that
V724R, C725S, A728S, S729T, D730N, and C731S channels had equal
susceptibilities to GIIIA and GIIIB (p > 0.05). In
contrast, although ~10-fold less sensitive to block by GIIIB than WT
Nav1.4, both N732K and N732R channels displayed GIIIA sensitivity not different from WT Nav1.4 (p > 0.05). Therefore, these constructs could discriminate between GIIIA
and GIIIB in a manner similar to the DII P-S6 lysine mutants. Likewise,
WT Nav1.1 could also discriminate between the two toxin
forms (IC50 for GIIIA = 290.6 ± 11.4 nM, n = 11). This discriminative ability of
WT Nav1.1, however, was lost in the B-to-M chimeric double mutant (IC50 for GIIIA = 52.8 ± 10.6 nM, n = 6).
View larger version (21K):
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Fig. 6.
Effects of the triple mutation
N732K/D762K/E765K on µ-CTX block. Top
panel, representative Na+ currents through
N732K/D762K/E765K channels in the absence and presence of 300 nM µ-CTX GIIIA (left) and 10 µM
GIIIB (right) as indicated. As anticipated from the
individual single mutations, N732K/D762K/E765K was insensitive to GIIIB
but sensitive to GIIIA. The effect of combining the individual DII
lysine mutations (N732K, D762K, and E765K) on GIIIB block was additive
(cf. Refs. 10 and 20). Bottom panel, a
typical experiment demonstrating the time course of the development of
onset and offset of GIIIA and GIIIB block of N732K/D762K/E765K during
toxin washin and washout as indicated. Normalized peak sodium currents
elicited by depolarization to 10 mV from a holding potential of
100
mV were plotted versus time in minutes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 kcal/mol when probed by
thermodynamic mutant cycle analysis) between the isoform-specific
sequence variants V724R, A728S, D730S (data not shown) and the
Arg13-Gln14-Lys16-Arg19
helical receptor-binding domain of µ-CTX which is known to interact prominently with the DII pore-lining segment of Nav1.4 (19, 20, 39). Based on these lines of reasoning, we hypothesize that the DII
S5-P determinants identified here may act as steric spacers that
allosterically modulate or control the access of other nearby
pore-lining residues (such as Glu758, Asp762,
and Glu765) that interact or are in direct contact with the
surface-blocking µ-CTX. This steric theory is consistent with the
dependence of toxin sensitivity on the side chain volume at position
728 and the lack of obvious electrostatic effects with the isoform
variants at position 730. During the preparation of this manuscript,
Cummins et al. (37) reported that GIIIB binding is
correlated inversely to the size of residue 729 (IC50 of
S729L > S729T > S729A
WT Nav1.4) (37).
Our findings that residue 732 modulates µ-CTX block and that the
toxin sensitivity follows N732D
WT Nav1.4 > N732R > N732K is also consistent with their results for a similar sensitivity sequence of WT Nav1.4
N732E
N732Q > N732K (37). Taken collectively, we propose a general
model for isoform-specific µ-CTX-channel interactions. When the DII
S5-P determinants are absent (or disrupted), the channel receptor in WT
Nav1.5 (or Nav1.1) becomes concealed so that
high affinity µ-CTX binding cannot occur despite the presence of all
other receptor constituents (such as Glu403,
Glu758, Asp762, Glu765,
Asp1241, and Asp1532 (8-10, 23) in the
conserved Na+ channel pore (Fig.
7). This "concealed receptor" theory
is similar to the "guarded receptor" theory (40) except that access
to the receptor is static rather than gated. It can be generalized to
explain other isoform-specific protein-ligand interactions. Although an
active site or a ligand receptor may be well conserved among different
isoforms of the same protein class, isoform-specific properties may
differ markedly because of surrounding regions that do not otherwise
participate directly in the biological activity in question. Because
these "scaffolds" are usually more variable among different
isoforms, they may represent unique sites for drug targeting whereby a
relatively nonselective drug moiety can be delivered to the nearby
conserved active site with imposed specificity as a result of the
increased local effective concentration around the active site caused
by its binding to the targeted scaffolds (41).
View larger version (32K):
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Fig. 7.
A schematic representation of
isoform-specific µ-CTX interactions with
Nav1.1 (left), 1.4 (middle), and 1.5 (right)
channels. When the Nav1.4 determinants in the DII S5-P
linker (red) are disrupted (in Nav1.1) or absent
(in Nav1.5), the µ-CTX receptor in the pore becomes
distorted or concealed such that optimal high affinity µ-CTX binding
cannot occur despite the presence of all other receptor constituents in
the P loops (purple) in these isoforms. The four
Na+ channel domains are arranged in a clockwise
configuration (19). The binding orientation of µ-CTX to
Nav1.4 is based on previously identified toxin-channel
interactions (19, 20, 21). The positions of Nav1.4
residues 729 and 732 (925 and 928 of Nav1.1) are only
approximate.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R01 HL-52768 (to E. M. and R. A. L.), R01 HL-50411 (to G. F. T.), and R01 NS-26729 (to A. L. G.).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.
§ Recipient of a research career development award from the Cardiac Arrhythmias Research and Education Foundation. To whom correspondence should be addressed: Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross 844, Baltimore, MD 21205. E-mail: ronaldli@jhmi.edu.
¶ Supported by a fellowship award from Universidad Nacional de La Plata, Argentina.
Present address: Facultad de Ciencias Médicas,
Universidad Nacional de La Plata, La Plata 1900, Argentina.
Recipient of the Michel Mirowski, M.D., professorship of
cardiology of The Johns Hopkins University.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M210882200
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ABBREVIATIONS |
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The abbreviations used are: µ-CTX, µ-conotoxin(s); B-to-M, brain-to-muscle; DI, DII, DIII, DIV, domains I, II, III, and IV, respectively; H-to-M, heart-to-muscle; STX, saxitoxin; TTX, tetrodotoxin; WT, wild-type.
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