From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the ¶ Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada, and the ** Departments of Medicine and Physiology, Emory University, Atlanta, Georgia 30322
Received for publication, December 1, 2000, and in revised form, January 10, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
µ-Conotoxins (µ-CTXs) specifically inhibit
Na+ flux by occluding the pore of voltage-gated
Na+ channels. Although the three-dimensional structures of
µ-CTXs are well defined, the molecular configuration of the channel
receptor is much less certain; even the fundamental question of whether the four homologous Na+ channel domains are arranged in a
clockwise or counter-clockwise configuration remains unanswered.
Residues Asp762 and Glu765 from domain
II and Asp1241 from domain III of rat skeletal muscle
Na+ channels are known to be critical for µ-CTX binding.
We probed toxin-channel interactions by determining the potency of
block of wild-type, D762K, E765K, and D1241C channels by
wild-type and point-mutated µ-CTXs (R1A, Q14D, K11A, K16A, and R19A).
Individual interaction energies for different toxin-channel pairs were
quantified from the half-blocking concentrations using mutant cycle
analysis. We find that Asp762 and Glu765
interact strongly with Gln14 and Arg19 but not
Arg1 and that Asp1241 is tightly coupled to
Lys16 but not Arg1 or Lys11. These
newly identified toxin-channel interactions within adjacent domains,
interpreted in light of the known asymmetric toxin structure, fix the
orientation of the toxin with respect to the channel and reveal that
the four internal domains of Na+ channels are arranged in a
clockwise configuration as viewed from the extracellular surface.
µ-Conotoxins
(µ-CTX)1 are receptor site
I sodium channel blockers (1) isolated from the sea snail Conus
geographus (2-5) that inhibit Na+ flux in
voltage-gated Na+ channels by physically occluding the pore
(2, 6-9). They are peptides consisting of 22 amino acid residues with
six cysteines that form three intramolecular disulfide bonds, imparting
backbone rigidity (2-7, 10-11). Their three-dimensional structures
are well defined (12-14), making µ-CTXs useful probes to study the Na+ channel pore structure. Unlike tetrodotroxin and
saxitoxin, whose smaller sizes render their receptor sites more compact
(15-20), high-affinity binding of µ-CTXs results from the summed
effects of numerous weaker toxin-channel interactions (9, 21-23). In contrast to charybdotoxin (ChTx) or agitoxin (AgTx), whose binding to
the homotetrameric K+ channels can assume any one of four
orientations with respect to the channel (24-26), µ-CTX binds to
Na+ channels presumably in only one (as yet undetermined)
favorable orientation because of intrinsic pore asymmetry (9, 18,
27-30). This notion is supported by the findings that critical channel residues for µ-CTX block identified to date are mostly clustered in
domain II (DII), while "homologous" residues in other domains often
have less or no effect (9, 30-32). Previously, Chang et al.
(32) demonstrated that the toxin residue Arg13, which is
required to inhibit current flow, interacts intimately with the DII
pore residue Glu758. Based on this observation, the
estimated dimensions of the channel pore, and the known structure of
the toxin, these authors proposed that µ-CTX docks like an inverted
pyramid with Arg13 reaching the deep pore region. The
experiments that we describe here were designed to determine the actual
µ-CTX docking orientation around the central axis.
Despite the detailed knowledge regarding the three-dimensional
structure of µ-CTX, the molecular configuration of its channel receptor remains uncertain. Unlike K+ channels, which
consist of four identical monomeric subunits, the pore-forming subunits
of Na+ and Ca2+ channels are single
polypeptides composed of four nonidentical homologous domains connected
by cytoplasmic linkers (Fig. 1A). Although various lines of
evidence have demonstrated that the four Na+ channel
domains are structurally asymmetrical (9, 18, 27-30), the fundamental
question of whether these domains are arranged in a clockwise or
counter-clockwise configuration (when viewed from the extracellular
side) remains unanswered (Fig. 1B). Indeed, review articles
from different laboratories continue to depict the channels in either
clockwise (33) or counterclockwise (1) topology, reflecting the paucity
of data favoring one model over the other.
The negatively charged residues Asp762 and
Glu765 located in the DII P-S6 linker (30) and
Asp1241 of the DIII P-loop (9) of the rat skeletal muscle
(µ1) Na+ channel are important for µ-CTX binding. These
DII and DIII channel residues are exposed to the aqueous phase of the
pore and influence single channel conductance (27, 29). Despite the
significant roles of these residues in µ-CTX binding, no site of
interaction on the toxin has been reported to date. Identification of
the interacting partners, on the toxin, of these DII and DIII channel residues will not only reveal the docking orientation of µ-CTX when
bound but will also help to define the internal domain arrangement (Fig. 1B). To identify the
toxin-channel interaction sites, we synthesized five GIIIA-based toxin
derivatives: R1A, K11A, Q14D, K16A, and R19A. These toxin mutants were
selected because their side chains protrude from different faces of the
toxin molecule (Fig. 2), enabling a
rigorous dissection of toxin interactions with the channel residues in
domains II and III. We first examined the IC50 for block of
wild-type (WT), D762K, E765K, and D1241C mutant channels by both WT and
point-mutated toxins and then applied mutant cycle analysis to
quantitate the individual interaction (or coupling) energies between
these channel and toxin sites. Our results indicate that
Asp762 and Glu765 interact with
Gln14 and Arg19, and Asp1241
interacts with Lys16. These results constrain the toxin
binding orientation and reveal that the four Na+ channel
domains are arranged in a clockwise configuration (when viewed from the
extracellular face).
Site-directed Mutagenesis and Heterologous Expression--
µ1
sodium channel (34) mutants were created using polymerase chain
reaction as previously described (29, 35). Na+ channels
were heterologously expressed in tsA-201 cells using the LipofectAMINE
Plus transfection kit (Life Technologies, Inc.). Transfected cells were
incubated at 37 °C in a humidified atmosphere of 95%
O2, 5% CO2 for 48-72 h for channel
protein expression before electrical recordings.
Synthesis of Point-mutated µ-CTX--
The GIIIA form of
µ-CTX was used in this study. Toxin derivatives were synthesized as
previously described (21, 32). Briefly, solid phase synthesis was
performed on a polystyrene-based Rink amide resin using
9-fluorenylmethoxycarbonyl chemistry. Synthesized peptides were
air-oxidized and purified by high pressure liquid chromatography.
Peptide composition was verified by quantitative amino acid analysis
and/or mass spectroscopy. One-dimensional proton NMR spectra of the
synthesized toxin were compared with those of native CTX to ensure
proper folding of the point-mutated toxin.
Electrophysiology and Data Analysis--
Electrophysiological
recordings were performed using the whole-cell patch clamp technique
(36) with an integrating amplifier (Axopatch 200A; Axon Instruments).
Transfected cells were identified by epifluorescence microscopy.
Pipette electrodes had a final tip resistances of 1-3 megaohms.
Recordings were performed at room temperature in a bath solution
containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
10 mM HEPES, 10 mM glucose, pH adjusted to 7.4 with NaOH. Only cells expressing peak Na+ currents between
1 and 5 nA were used to ensure good current resolution while
maintaining adequate voltage control. Designated toxin amounts were
added to the bath when required. The internal recording solution
contained 35 mM NaCl, 105 mM CsF, 1 mM MgCl2, 10 mM HEPES, 1 mM EGTA, pH adjusted to 7.2 with CsOH. All chemicals were
purchased from Sigma unless otherwise specified.
Toxin was superfused continuously during the experiment. Flow rate was
maintained at ~5 ml/min (bath volume was 150 µl). Wash-out began
only after the peak currents had reached a steady-state level. Raw
current records were analyzed using custom-written software.
Equilibrium half-blocking concentrations (IC50) for µ-CTX
were determined from the following binding isotherm,
For mutant cycle analysis, coupling/interaction energies
(
All data are reported as mean ± S.E. Statistical significance was
determined using a paired Student's t test at the 5% level.
Molecular Interactions between µ-CTX and the DII P-S6 Residues
Asp762 and Glu765--
We initially looked for
molecular interactions between the DII P-S6 channel residues
(i.e. Asp762 and Glu765) and µ-CTX
variants using mutant cycle analysis. Prediction of interactions
(attractive or repulsive) between channel and toxin residues using this
analysis requires determinations of blocking affinity of all of the
following combinations: WT channel-WT toxin, mutant channel-WT toxin,
WT channel-mutant toxin, and mutant channel-mutant toxin (see
"Experimental Procedures" and Ref. 37 for review). Therefore, we
first determined the half-blocking concentrations (IC50
values) for block of WT µ1 Na+ channels by both WT
µ-CTX and the mutant toxin derivatives R1A, Q14D, and R19A. Fig.
3A shows representative
whole-cell sodium currents recorded from cells expressing WT channels
in the absence and presence of the toxin variants. The corresponding
dose-response curves for binding of these toxins are shown in Fig.
3B. Consistent with previous results (11, 13, 21), the toxin
derivatives R1A, Q14D, and R19A displayed significantly reduced
affinity for block of WT Na+ channels compared with WT
µ-CTX. Using the same strategy, we then determined the
IC50 for block of the DII P-S6 mutants D762K and E765K by
the same toxins. The IC50 values for these toxin-channel pairs are summarized in Fig. 4. Whereas
both D762K and E765K channels were blocked by WT or R1A toxins with
reduced potency, their block by Q14D and R19A was augmented compared
with WT channels (Fig. 4). From the measured IC50 values,
we estimated coupling or interaction energies ( Electrostatic Interactions between µ-CTX and
Asp1241--
We and others have shown that neutralization
of the charge on the DIII P-loop residue Asp1241
significantly destabilizes µ-CTX binding (9, 32, 38). However, the
role of this residue in µ-CTX's interactions with the
Na+ channel is undefined. Since µ-CTX carries a net
positive charge and Asp1241 is negatively charged and
exposed to the aqueous pore (9, 27), the toxin and this channel residue
may interact electrostatically. To define the toxin interaction with
this residue, we introduced the point mutations D1241C for charge
neutralization, D1241K for charge reversal, and D1241E for charge
conservation; the effects on µ-CTX block are summarized in Fig.
6. Consistent with previous results (9),
D1241C (IC50 = 3.3 ± 1.5 µM,
n = 3) exhibited reduced block by µ-CTX compared with
WT channels. As expected for an electrostatic effect, the
charge-reversed mutation D1241K (IC50 = 9.4 ± 3.4 µM, n = 3) further destabilized toxin
affinity compared with the charge-neutralized cysteine mutant. In
contrast, the charge-conserving mutation D1241E partially preserved
sensitivity to µ-CTX (IC50 = 110.0 ± 20.1 nM, n = 4); D1241E channels were only
~3-fold less sensitive than WT channels. Taken together, these
results support the notion that electrostatic interactions exist
between Asp1241 and µ-CTX.
Interaction of Asp1241 with Lys16--
To
further constrain the binding orientation, we studied the interactions
of DIII-D1241 with µ-CTX, looking for couplings between this channel
residue and Arg1, Lys11, and Lys16
of the toxin. These toxin sites were chosen because their side chains
protrude from different faces of µ-CTX compared with
Gln14 and Arg19 (see Fig. 2 for locations of
these residues). Fig. 7A shows
the IC50 values for block of D1241C channels by the µ-CTX
derivatives R1A, K11A, and K16A. Interestingly, the potency of block by
K16A was significantly greater in D1241C channels than in WT channels, consistent with a stronger interaction between the D1241C mutant channel and K16A than between WT channels and K16A. This change in
interaction energy could reflect a gain of attractive and/or a relief
of repulsive forces, leading to increased potency of the K16A
derivative. In contrast, the potencies of block by either R1A or K11A
were reduced when applied to D1241C channels, in a similar manner to
that seen with the WT channels, indicating an additive effect of these
channel and toxin mutations. Fig. 7B shows the coupling
energies for D1241C with R1A, K11A, and K16A. D1241C displays an
exceptionally strong positive interaction with Lys16
(3.2 ± 0.2 kcal/mol), implying a close association between these toxin and channel sites in the toxin-channel complex. Less significant interactions were identified with Arg1 (0.5 ± 0.2 kcal/mol) and with Lys11 (0.7 ± 0.2 kcal/mol).
µ-Conotoxin Macrosite--
Consistent with previous studies, our
experiments with alanine-substituted µ-CTX GIIIA derivatives on WT
µ1 channels show significant reductions in toxin-binding affinity,
suggesting that the positively charged toxin residues Arg1,
Lys11, Lys16, and Arg19 are
critical for its biological activity. These results were qualitatively
similar to previous studies using either glutamine or alanine
substitution for charge neutralization at these toxin positions, when
assessed using a rat diaphragm bioassay or bilayer-incorporated single
Na+ channels, respectively (11, 21). The reduction in
blocking affinity reported in these earlier studies, and in our own
data, was in the order Arg19 > Lys16 > Arg1 ~ Lys11. Since the side chains of these
toxin residues are spread over a wide surface and protrude in different
directions around the toxin molecule, these results are most consistent
with the notion that the µ-CTX binding site, unlike the ones for
tetrodotroxin and saxitoxin, covers a more extensive area of the
channel surface. Additionally, the highly site-specific pattern of
interactions among the various toxin and channel sites further implies
that intimate toxin-channel interactions are localized and argue
against global disruptions of either the channel or the toxin by the
mutations introduced.
Charge manipulations at position 1241 in DIII by point mutations
support an electrostatic role for this residue in µ-CTX binding, since the charge reversal mutation (D1241K) further destabilized toxin
block compared with the charge-neutralized mutant (D1241C), whereas
conservation of charge (D1241E) partially restored wild-type toxin
affinity. This incomplete restoration by the glutamate substitution suggests that an aspartate is required at position 1241 to confer wild-type µ-CTX blocking behavior. This observation could be
attributed to steric hindrance, since the glutamate side chain is one
methyl group longer than that of aspartate. Overall, the data suggest that the charges, as well as their precise location within the pore,
are critical for toxin-channel interaction.
The
Arg13-Gln14-Lys16-Hyp17-Arg19
Toxin Face Recognizes the Channel Receptor--
Inspection of the
three-dimensional structures of GIIIA µ-CTX reveals that
Arg13, Gln14 (or Arg14 for µ-CTX
GIIIB, whose three-dimensional structure is essentially the same as
GIIIA) (14), Lys16, Hyp17, and
Arg19 are clustered on one helical face of the toxin
molecule (Fig. 2, bottom panel) (11-14). We and
others have demonstrated that Arg13, Lys16,
Hyp17, and Arg19 are essential residues for the
biological activity of the toxin (see Refs. 11, 13, 21, 32, 39, and 40,
and this report). Here we further show that Gln14 and
Arg19 of the toxin interact strongly with the DII P-S6
channel residues Asp762 and Glu765. Previously,
Chang et al. (32) showed that Arg13 associates
closely with the DII P-loop residue Glu758. However, even
with the assumption that Arg13 is directed inward,
approximately parallel to the conducting axis of the pore, these
findings still do not completely define the docking orientation of
µ-CTX with respect to all channel domains, because the data are
limited to toxin-domain II interactions. We therefore studied also the
interactions of DIII-D1241 with the toxin. Our results indicate that
Asp1241 interacts strongly with Lys16 but not
Arg1 and Lys11 (Fig. 7). Taken together, these
findings highly suggest that the toxin
Arg13-Gln14-Lys16-Hyp17-Arg19
helical face interacts with the channel receptor site (13-14). Fig.
8 shows a schematic representation of a
possible docking orientation of µ-CTX based on the pattern of
specific toxin-channel interactions observed. According to this model,
the
Arg13-Gln14-Lys16-Hyp17-Arg19
helical face associates closely with the P-segment (including the P-S6
linker) of domain II. Arg13 projects into the pore and is
closest to Glu758. Asp762 and
Glu765 both closely interact with Gln14 as well
as Arg13 but are capable of interacting also with
Arg19. The proposed docking orientation of µ-CTX predicts
that Arg1 faces the opposite direction and is remote from
DII, consistent with our experimental observations (cf. Fig.
5). In addition, our model also predicts that Lys11
protrudes in a direction away from the DII P-loop and therefore, like
R1, also should not interact strongly with any of the DII residues.
Clockwise Domain Arrangement--
The strong coupling between the
toxin residue Lys16 and the channel residue
Asp1241 in DIII (Fig. 7) place these toxin and channel
residues in close proximity with each other in the toxin-bound state.
According to the toxin orientation proposed above, Lys16
lies above Arg13, implying that Asp1241 is
located shallower within the pore than Glu758. This is
consistent with fractional electrical distances estimated from single
channel recordings (27). This arrangement of the toxin molecule,
channel domains, and residues also explains the somewhat marginal
interaction of Asp1241 with Lys11, since this
toxin residue may not directly face Asp1241 (or DIII).
Based on the known structure of µ-CTX and the proposed toxin docking
orientation, our results suggest a clockwise arrangement of the channel
domains when viewed from the external surface (Fig. 8). This conclusion
is consistent with that proposed recently by Dudley et al.
(39), although in general their conclusion was based on entirely
different lines of evidence and interpretation. The models of
toxin-channel docking in the two studies differ in several important
details. In contrast to our model, Dudley et al. place
Lys16 between DI and DIV. They detected little binding
energy between Lys16 and Met1240 in DIII using
the mutant pair K16A and M1240A and propose that Arg1 is
adjacent to DII using R1A and T759I. Their arguments were based on
analysis of possible interactions with all four domains, which, in
general, were energetically weaker than the key interactions reported here.
Conclusion--
In summary, we have demonstrated that the toxin
residues Gln14 and Arg19 interact with the DII
P-S6 channel residues Asp762 and Glu765 and
that Lys16 interacts strongly with DIII-D1241. These data
suggest a unique docking orientation of µ-CTX. Based on these results
and the known three-dimensional structure of µ-CTX, we further
conclude that the four Na+ channel domains are arranged in
a clockwise configuration when viewed from the extracellular side.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (57K):
[in a new window]
Fig. 1.
A, schematic representation of the
putative transmembrane topology of the Na+ channel
-subunit. The regions between the fifth and sixth transmembrane
segments (S5 and S6) form the P-loops. The approximate location of the
DII P-S6 linker (thick red lines)
where Asp762 and Glu765 are located is shown.
Asp1241 (red star) is located in the
ascending limb of the DIII P-loop. B, schematic
representation of a clockwise (left panel) and a
counter-clockwise (right panel) domain
arrangement of the four homologous domains of the Na+
channel as viewed from the extracellular side. Identification of
toxin-channel interacting points in different domains will allow
determination of the correct configuration of domain arrangement.
Suppose residue A (red triangle) of the blocker
is known to interact with DII; evidence showing interaction of residue
B (cyan triangle) with DIII would suggest a
clockwise domain arrangement. In contrast, interactions of DIII with
residue C (purple triangle) would indicate a
counter-clockwise arrangement.
View larger version (25K):
[in a new window]
Fig. 2.
Three-dimensional structure of
µ-CTX-GIIIA. Top panel, toxin
residues studied in this report (i.e. Arg1,
Lys11, Gln14, Lys16, and
Arg19) and Arg13 are indicated with one-letter
amino acid codes. Arg13 has been demonstrated to interact
strongly with the DII pore residue Glu758. Since the side
chains of these toxin residues protrude from different faces of the
toxin, identification of novel channel/toxin contact points will reveal
information regarding the orientation of µ-CTX when it is bound to
the channel, thereby providing insights to the pore structure.
Bottom panel, µ-CTX displayed in ribbon format.
The helical face consisting of Arg13, Gln14,
Lys16, Hyp17, and Arg19 is
highlighted (magenta).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
where IC50 represents the half-blocking
concentration, and IO 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 toxin, respectively.
G) for various mutant toxin-channel pairs were
calculated from the equilibrium IC50 values using the
following equations,
(Eq. 2)
(Eq. 3)
where R is the gas constant and T is the
Kelvin temperature. The S.E. values for
(Eq. 4)
G were
estimated by dividing the square root of the sum of the variances of
the RT ln IC50 means by the square root of the
number of degrees of freedom.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G)
between these toxin and channel sites (Fig.
5). In general, the patterns of
interactions for D762K and E765K were similar. Both DII-PS6 residues
had the strongest interactions with Q14D (D762K/Q14D = 1.5 ± 0.1 kcal/mol; E765K/Q14D = 1.3 ± 0.1 kcal/mol), followed by
R19A (D762K/R19A = 0.9 ± 0.1 kcal/mol; E765K/R19A = 1.0 ± 0.1 kcal/mol); in contrast, using the conventional cut-off
of 0.5 kcal/mol for significance (38), there was no clear interaction
with R1A (D762K/R1A = 0.5 ± 0.1 kcal/mol; E765K/R1A = 0.5 ± 0.1 kcal/mol). These results might be explained if
Asp762 and Glu765 are close to
Gln14, farther from Arg19, and distant from
Arg1 in the toxin-bound state.
View larger version (20K):
[in a new window]
Fig. 3.
A, representative raw current traces of
WT channels elicited by depolarization to 10 mV from a holding
potential of
100 mV in the absence and presence of WT µ-CTX, R1A,
Q14D, or R19A as indicated. Control peak amplitudes were normalized for
comparison. Their magnitudes were 2.6, 4.9, 2.6, and 5.0 nA for WT
GIIIA, R1A, Q14D, and R19A, respectively. B, the
dose-response relationship for block of WT channels by WT µ-CTX and
its derivatives. Normalized peak Na+ currents at
10 mV
were plotted as a function of extracellular µ-CTX concentrations.
Data points were fitted with a binding isotherm to estimate the
IC50 for µ-CTX block of each channel (see "Experimental
Procedures"). Data are plotted as mean ± S.E.
View larger version (48K):
[in a new window]
Fig. 4.
A, representative sodium currents from
D762K and E765K channels recorded in the absence and presence of WT,
R1A, Q14D, or R19A µ-CTX as indicated. Control peak amplitudes for
D762K channels were 2.6, 2.6, 4.1, and 3.6 nA for block by WT CTX, R1A,
Q14D, and R19A, respectively. For E765K channels, they were 2.9, 3.1, 5.0, and 1.9 nA, respectively. B, bar graphs summarizing the
half-blocking concentrations (IC50) for block of WT, D762K,
and E765K channels by WT and mutant µ-CTX derivatives R1A, Q14D, and
R19A. Data presented were averaged data from 3-10 individual
determinations.
View larger version (18K):
[in a new window]
Fig. 5.
Estimated interaction energies
( G) of D762K and E765K with R1A,
Q14D, and R19A. Q14D and R19A but not R1A interact significantly
(see text) with the DII P-S6 residues D762K and E765K.
Interactions of D762K and E765K were stronger with Q14D than with R19A,
suggesting that these channel residues are more closely associated with
Gln14 than with Arg19 when the toxin molecule
is bound to the channel.
View larger version (15K):
[in a new window]
Fig. 6.
Effects of charge neutralization, reversal,
and restoration at position DIII-D1241 on block by
µ-CTX. A, representative
Na+ current tracings of WT, D1241C, D1241K, and D1241E
channels elicited by depolarization to 10 mV from a holding potential
of
100 mV in the absence and presence of µ-CTX as indicated by the
arrows. Toxin-free peak currents were normalized for ease of
comparison. Their magnitudes were 2.6, 4.2, 3.2, and 3.5 nA,
respectively. B, bar graphs summarizing the half-blocking
concentrations (IC50) for µ-CTX of WT and the same
channels shown in A. D1241C displayed a ~100-fold
reduction in µ-CTX affinity compared with WT channels. The
charge-reversed mutation D1241K further destabilized toxin binding. The
charge-conserved mutation D1241E only partially restored wild-type
µ-CTX sensitivity. Its IC50 was 3-fold higher than WT.
Data are all presented as mean ± S.E.
View larger version (33K):
[in a new window]
Fig. 7.
Interactions of D1241C with Arg1
and Lys16. A, bar graphs
summarizing the IC50 values of WT (open
bars) and D1241C channels (closed
bars) for block by R1A, K11A, and K16A. Data presented are
mean ± S.E. B, interaction energies
( G) of D1241C with R1A, K11A, and K16A calculated from
the experimental IC50 values shown in A. D1241C
displays a substantial interaction with K16A and a marginal interaction
with K11A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (42K):
[in a new window]
Fig. 8.
Clockwise arrangement of channel
domains. A graphical representation is shown for the interactions
between µ-CTX, the DII P-loop, and DIII D1241 when bound to the
Na+ channel pore. The model depicts the helical face of
µ-CTX, which comprises Arg13, Gln14,
Lys16, Hyp17, and Arg19,
interacting closely with the critical DII channel residues
Glu758, Asp762, and Glu765. The
back bone loop and placement of residues Asp762 and
Glu765 are generally consistent with the electrical
distance data of Li et al. (29), but there are no
specific data that constrain the turn in the DII backbone to be either
right- or left-handed. In the actual channel-toxin complex, side chain
configurations may differ from those in the solution structure, adding
some uncertainty to the predictions of positions of pore-lining
residues. Arg1 and Lys11 face different sides
of the channel (cf. Fig. 2) and do not interact with DII.
DIII-D1241 associates closely with Lys16. The channel
domains are arranged in a clockwise configuration when viewed from the
extracellular surface.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 HL-52768 (to E. M.) and R01 HL-50411 (to G. F. T.) and by operating funds from the Medical Research/Canadian Institutes of Health Research (to R.J.F.).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 fellowship award from the Heart and Stroke
Foundation of Canada.
§ Supported by a fellowship award from Universidad Nacional de La Plata, Argentina.
Recipient of salary support as a Medical Research
Council/Canadian Institutes of Health Research Distinguished Scientist
and an Alberta Heritage Foundation for Medical Research Medical Scientist.
Recipient of salary support from a Scientist Development Award
from the American Heart Association and National Institutes of Health
Grant HL-64828.
§§ Holder of the Michel Mirowski, M.D. Professorship of Cardiology of The Johns Hopkins University. To whom correspondence should be addressed: Inst. of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave./Ross 844, Baltimore, MD 21205. Tel.: 410-955-2776; Fax: 410-955-7953; E-mail: marban@jhmi.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010862200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: µ-CTX, µ-conotoxin; WT, wild-type; DII, DIII, and DIV, domain II, III, and IV, respectively; Hyp, hydroxyproline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Catterall, W. A. (1988) Science 242, 50-61[Medline] [Order article via Infotrieve] |
2. |
Cruz, L. J.,
Gray, W. R.,
Olivera, B. M.,
Zeikus, R. D.,
Kerr, L.,
Yoshikami, D.,
and Moczydlowski, E.
(1985)
J. Biol. Chem.
260,
9280-9288 |
3. | Gray, W. R., Olivera, B. M., and Cruz, L. J. (1988) Annu. Rev. Biochem. 57, 665-700[CrossRef][Medline] [Order article via Infotrieve] |
4. | Olivera, B. M., Rivier, J., Clark, C., Ramilo, C. A., Corpuz, G. P., Abogadie, F. C., Mena, E. E., Woodward, S. R., Hilliyard, D. R., and Cruz, L. J. (1990) Science 249, 257-263[Medline] [Order article via Infotrieve] |
5. | Nakamura, H., Kobayashi, J., Ohizumi, Y., and Hirata, Y. (1983) Experientia (Basel) 39, 590-591 |
6. | Moczydlowski, E., Olivera, B. M., Gray, W. R., and Strichartz, G. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5321-5325[Abstract] |
7. | Yanagawa, Y., Abe, T., and Satake, M. (1986) Neurosci. Lett. 64, 7-12[Medline] [Order article via Infotrieve] |
8. | Dudley, S. C., Jr., Hannes, T., Lipkind, G., and Fozzard, H. A. (1995) Biophys. J. 69, 1657-1665[Abstract] |
9. | Li, R. A., Tsushima, R. G., and Backx, P. H. (1997) Biophys. J. 73, 1874-1884[Abstract] |
10. | Hidaka, Y., Sato, K., Nakamura, H., Ohizumi, Y., Kobayashi, J., and Shimonishi, Y. (1990) FEBS Lett. 264, 29-32[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Sato, K.,
Ishida, Y.,
Wakamatsu, K.,
Kato, R.,
Honda, H.,
Ohizumi, Y.,
Nakamura, H.,
Ohya, M.,
Lancelin, J. M.,
Kohda, D.,
and Inagaki, F.
(1991)
J. Biol. Chem.
266,
16989-16991 |
12. | Lancelin, J. M., Knoda, D., Tate, S., Yanagawa, Y., Abe, T., Satake, M., and Inagaki, F. (1991) Biochemistry 30, 6908-6916[Medline] [Order article via Infotrieve] |
13. | Wakamatsu, K., Kohda, D., Hatanaka, H., Lancelin, J. M., Ishida, Y., Oya, M., Nakamura, H., Inagaki, F., and Sato, K. (1992) Biochemistry 31, 12577-12584[Medline] [Order article via Infotrieve] |
14. | Hill, J. M., Alewood, P. F., and Craik, D. J. (1996) Biochemistry 35, 8824-8835[CrossRef][Medline] [Order article via Infotrieve] |
15. | Backx, P., Yue, D., Lawrence, J., Marbán, E., and Tomaselli, G. (1992) Science 257, 248-251[Medline] [Order article via Infotrieve] |
16. | Heinemann, S. H., Terlau, H., and Imoto, K. (1992) Pflügers Arch. 422, 90-92[Medline] [Order article via Infotrieve] |
17. | Satin, J., Kyle, J. W., Chen, M., Bell, P., Cribbs, L. L., Fozzard, H. A., and Rogart, R. B. (1992) Science 256, 1202-1205[Medline] [Order article via Infotrieve] |
18. |
Perez-Garcia, M. T.,
Chiamvimonvat, N.,
Marbán, E.,
and Tomaselli, G. F.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
300-304 |
19. | Terlau, H., Heinemann, S. H., Stühmer, W., Pusch, M., Conti, F., Imoto, K., and Numa, S. (1991) FEBS Lett. 293, 93-96[CrossRef][Medline] [Order article via Infotrieve] |
20. | Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13[Abstract] |
21. | Becker, S., Prusak-Sochazewski, E., Zamponi, G., Beck-Sickinger, A. G., Gordon, R. D., and French, R. J. (1992) Biochemistry 31, 8229-8238[Medline] [Order article via Infotrieve] |
22. | Stephan, M. M., Potts, J. F., and Agnew, W. S. (1994) J. Membr. Biol. 137, 1-8[Medline] [Order article via Infotrieve] |
23. | Chen, L.-Q., Chahine, M., Kallen, R. G., Barchi, R. L., and Horn, R. (1992) FEBS Lett. 309, 253-257[CrossRef][Medline] [Order article via Infotrieve] |
24. | MacKinnon, R., and Miller, C. (1989) Science 245, 1382-1385[Medline] [Order article via Infotrieve] |
25. | Goldstein, S. A. N., Pheasant, D. J., and Miller, C. (1994) Neuron 12, 1377-1388[Medline] [Order article via Infotrieve] |
26. | Gross, A., and MacKinnon, R. (1996) Neuron 16, 399-406[Medline] [Order article via Infotrieve] |
27. | Chiamvimonvat, N., Perez-Garcia, M., Ranjan, R., Marbán, E., and Tomaselli, G. F. (1996) Neuron 16, 1037-1047[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Benitah, J. P.,
Tomaselli, G. F.,
and Marbán, E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7392-7396 |
29. |
Li, R. A.,
Velez, P.,
Chiamvimonvat, N.,
Tomaselli, G. F.,
and Marbán, E.
(2000)
J. Gen. Physiol.
115,
81-92 |
30. |
Li, R. A.,
Ennis, I.,
Velez, P.,
Tomaselli, G. F.,
and Marbán, E.
(2000)
J. Biol. Chem.
275,
27551-27558 |
31. |
Chahine, M.,
Sirois, J.,
Marcotte, P.,
Chen, L.-Q.,
and Kallen, R. G.
(1998)
Biophys. J.
75,
236-246 |
32. | Chang, N. S., French, R. J., Lipkind, G. M., Fozzard, H. A., and Dudley, S., Jr. (1998) Biochemistry 37, 4407-4419[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Marbán, E.,
Yamagishi, T.,
and Tomaselli, G. F.
(1998)
J. Physiol.
508,
647-657 |
34. | Trimmer, J. S., Cooperman, S. S., Tomiko, S. A., Zhou, J., Crean, S. M., Boyle, M. B., Kallen, R. G., Sheng, Z., Barchi, R. L., Sigworth, F. J., Goodman, R. H., Agnew, W. S., and Mandel, G. (1989) Neuron 3, 33-49[Medline] [Order article via Infotrieve] |
35. | Yamagishi, T., Janecki, M., Marbán, E., and Tomaselli, G. F. (1997) Biophys. J. 73, 195-204[Abstract] |
36. | Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100[Medline] [Order article via Infotrieve] |
37. | French, R. J., and Dudley, S. C., Jr. (1999) Methods Enzymol. 294, 575-605[Medline] [Order article via Infotrieve] |
38. | Schreiber, G., and Fersht, A. R. (1995) J. Mol. Biol. 248, 478-486[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Dudley, S. C., Jr.,
Chang, N.,
Hall, J.,
Lipkind, G.,
Fozzard, H.,
and French, R. J.
(2000)
J. Gen. Physiol.
116,
679-690 |
40. | Chahine, M., Chen, L.-Q., Fotouhi, N., Walsky, R., Fry, D., Horn, R., and Kallen, R. G. (1995) Recept. Channels 3, 164-74 |