From the Department of Crystallography, Birkbeck
College, University of London, London WC1E 7HX, United Kingdom and
¶ Unité Mixté de Recherche 6026 CNRS-Université
de Rennes I, Laboratoire des Interactions Cellulaires et
Moléculaires, Campus de Beaulieu, Batiment 13, 35042 Rennes Cedex, France
Received for publication, August 15, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Voltage-gated sodium channels are dynamic
membrane proteins characterized by rapid conformational changes that
switch the molecule between closed resting, activated, and inactivated
states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the
inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that
causes steady-state activation and binds in the inner pore of the
sodium channel with overlapping but distinct molecular determinants
from those of lamotrigine. Using circular dichroism spectroscopy on
purified voltage-gated sodium channels from Electrophorus
electricus, the secondary structures associated with the mixture
of states present at equilibrium in the absence of these ligands were
compared with specific stabilized states in their presence. As the
channel shifts to open states, there appears to be a significant change
in secondary structure to a more Voltage-gated sodium channels play an important physiological role
in excitable membranes, underlying action potential initiation and
propagation in nerves and muscles (1). They are also involved in a
number of pathophysiologies, e.g. rhythm dysfunctions in the
heart (2), and channelopathies (3, 4) due to inherited mutations,
including hyperkaleimic periodic paralysis, myotonia congenita, and
Long QT syndrome. Although their functions have been extensively
characterized via electrophysiology (5), structural studies remain
scanty, and the molecular basis of the central process of gating is
still elusive despite recent progress (6).
Sodium channels show strong sequence conservation across species and
tissue-specific types (7). The sodium channel from the electric eel
electroplax is 60% identical with that of voltage-gated sodium
channels from human muscle. The patterns of hydrophobicity and
homologous residues are even more closely preserved, and this indicates
that their three-dimensional structures will be very similar. The
primary structure of the sodium channel from Electrophorus electricus was deduced from its cDNA sequence (8) and revealed the protein to consist of 1820 amino acids, producing a molecular mass of 208 kDa. There are also extensive sugar moieties
covalently linked to the protein on its external face, making the total
molecular mass of the channel ~260-270 kDa (9). Its sequence
contains four highly homologous internal repeats (domains I-IV), each
of which consists of six transmembrane-spanning segments (S1-S6), plus linking regions of differing lengths between the domains and
extended aqueous-soluble N- and C-terminal domains.
The intracellular linker between domains III and IV has been identified
by mutation studies (10) as being involved in fast inactivation, and an
NMR study of this peptide in isolation (11) suggested that it has the
potential for folding as an The low resolution (19 Å) three-dimensional structure of the sodium
channel from E. electricus determined by cryoelectron microscopy and single particle image analysis (16) revealed an overall
architecture of four domains with pseudo-4-fold symmetry surrounding a
central pore. The molecule is bell-shaped with an extracellular domain
and transmembrane domain together comprising ~50% of the total
volume and a large intracellular domain occupying the remaining volume.
The sodium channel can adopt distinct but correlated functional
conformational states during resting, activation, inactivation, and
deactivation. A conformational change upon voltage activation allosterically modifies the conformation of the transmembrane pore,
which is spatially separate from the voltage sensors (17). The sodium
channel acts as a capacitor in that there is a time lag between
stimulation and opening of sodium gates. The stimulus charges the
channel over a period of time leading to a conformational change or
sequence of conformational changes of the sodium channel protein, which
effectively opens the gate initiating an ionic current.
Neurotoxins and drugs such as local anesthetics, anticonvulsants, and
antiarrhythmics that modify sodium channel function have been shown to
bind preferentially to specific conformational states affecting either
activation, inactivation, or both (for a review, see Ref. 18). The
antiepileptic drug lamotrigine (LTG) binds preferentially to open
sodium channels during inactivation with apparent pore block (19).
Batrachotoxin (BTX) binds preferentially to the open state causing
steady-state activation (20), prevents all forms of inactivation, and
slows deactivation. LTG and BTX bind to specific sites that have been
shown by mutagenesis to have overlapping but distinct molecular
determinants in the S6 transmembrane segments of domains I, III, and IV
(19). Ligand binding appears to be facilitated by gating movements of
the pore segments allowing access to their binding sites (21).
To investigate the structural nature of the different functional states
of the sodium channel, we have used LTG and BTX to shift the
equilibrium mixture of states normally present in the absence of
ligands to one of the specific functionally defined conformational
states. CD spectroscopy was used to examine conformational changes
associated with binding of these ligands to the sodium channel
purified from E. electricus. These studies have shown that
when either lamotrigine or batrachotoxin binds there is a significant
change in secondary structure.
Genapol C-100 (10% solution) was obtained from Calbiochem. IgM
anti- Isolation and Solubilization of Electroplax Membranes
Membranes were purified from frozen electroplax tissue in a
manner similar to that previously described (23, 24) but with a number
of modifications designed to increase the yield and maintain protein
integrity: thawed diced tissue was suspended in 5 volumes of buffer A
(200 mM NaCl, 10 mM sodium phosphate buffer, pH
7.4, 5 mM EDTA, 1 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride, 0.025% pepstatin, 0.025%
leupeptin, 0.025% aprotinin, 0.02% NaN3) and homogenized
at 20,000 rpm for 1 min. After filtration, the sample was centrifuged
at 4000 × g for 30 min. The pellets were resuspended in 5 volumes of buffer A, and the homogenization, filtration, and
centrifugation steps were repeated. The pellets were resuspended in the
buffer at a concentration of 1.5 mg/ml and stored at Solubilization of proteins from electroplax membranes was achieved by
addition of Genapol C-100 to the membrane suspension, resulting in a
final detergent concentration of 2%. The suspension was homogenized
and centrifuged at 100,000 × g for 1 h at
2 °C. The supernatant contained solubilized electroplax membranes.
Immunoaffinity Chromatography Purification of Sodium
Channels
IgM was purified from an ammonium sulfate precipitate using an
affinity column of polyacrylhydrazido-agarose resin. Purified IgM was
coupled to CNBr-activated Sepharose 4B as previously described (24) at
ratios of 1.35 mg of IgM per ml of resin. The IgM affinity resin was
equilibrated with buffer B (50 mM sodium phosphate buffer, pH 7.4, 0.1% Genapol C-100, 0.2 mg/ml egg phosphatidylcholine, 5 mM EDTA, 1 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride, 0.025% pepstatin, 0.025% leupeptin,
0.025% aprotinin). The solubilized electroplax membrane fraction was
bound to the resin for 2 h. The protein was eluted using a linear
gradient of buffer B supplemented with 3.0 M KCl. The
eluted fractions were concentrated in a pressure ultrafiltration cell
(Amicon) with a PBMK 300K membrane (Millipore).
Reconstitution into Liposomes and Mixed Lipid Micelles
For electrophysiology studies, the sodium channel was
reconstituted into liposomes by dialysis of the protein solution
(concentration ~ 50 µg/ml) overnight against 1 liter of 150 mM sucrose, 0.5 mM MgCl2, 0.1 M Tris, and 25 mM HEPES, pH 7.4, using a
Spectra/Por membrane (molecular mass cut-off, 100 kDa). For CD
spectroscopy, the protein was dialyzed against 20 mM sodium
phosphate buffer containing 0.1% Genapol C-100 to produce
lipid/detergent mixed micelles.
Characterization of Purified Channels
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analyses--
The solubilized and purified protein fractions were
examined by SDS-polyacrylamide gel electrophoresis after the method of Laemmli (25). The gel was calibrated using standards ranging from 53 to
220 kDa. The 4-15% gradient PHAST acrylamide gels were silver-stained
(26), or the protein was electrotransferred to a polyvinylidene
difluoride membrane. For Western blot analysis, the primary antibody
was an aliquot of the purified anti-sialic acid horse IgM. The
secondary antibody was rabbit anti-horse IgG conjugated to alkaline phosphatase.
Protein Concentration Determination--
The relative protein
concentrations were determined with the bicinchoninic acid assay (27)
using bovine serum albumin as the standard. The bicinchoninic acid
analysis was calibrated for the absolute sodium channel protein
concentration via duplicate quantitative amino acid analyses of the
channel protein.
Single-channel Conductance in Planar Lipid Bilayers--
For
single channel conductance studies, planar lipid bilayers were formed
using the tip-dip method, (28) i.e. at the tip of a
patch-clamp pipette. A 1-ml Teflon chamber and the patch pipette were
filled with electrolytic solution (0.5 M NaCl, 10 mM HEPES buffer, pH 7.4, sterile filtered through 22 µm).
The pipette solution, equivalent to the cis-side of the
bilayer, was supplemented with 1 µM BTX. Ag/AgCl
electrodes were employed in the bath and pipette.
Bilayers were formed using the droplet technique, where 20 µl of a
bacterial phosphatidylethanolamine/bovine phosphatidylserine/egg phosphatidylcholine (5:4:1) lipid solution (1 mg/ml in hexane) were
applied to the micropipette shank just dipping at the air-salt solution
interface. A triangular voltage waveform of amplitude ~20 mV and
frequency ~100 Hz was applied to check capacitance values after
bilayer formation. After checking bilayer stability and electrical
silence under applied voltage, 20 µl of ~50 µg/ml reconstituted
sodium channels were added to the bath, i.e. the trans-side. The pipette electrode was connected to the
patch-amplifier through a preamplifier or headstage with a 1-giga-ohm
resistor (or gain). The sampling rate was 3 kHz, and the low-pass
Bessel filter was set at 300 Hz during data acquisition.
Electron Microscopy--
Aliquots of samples to be used for CD
spectroscopy were applied at a protein concentration of ~50 µg/ml
to carbon-coated copper grids and stained with 2% (w/v) uranyl
acetate. These were then examined with a TECNAI 1200Ex transmission
electron microscope operating at 120 kV.
Circular Dichroism Spectroscopy--
The CD spectra were
obtained using an Aviv 215 spectropolarimeter, which was specially
modified to have a large angle detection geometry (
The sodium channel protein (0.53 µM) was examined in the
presence and absence of ligands, either lamotrigine (50 µM) or BTX (1.5 µM). To each sample, ligand
dissolved in ethanol was added to produce a final ethanol concentration
of 5%. Ethanol was added to the baselines in the same amount. An
equivalent amount of ethanol was added to samples without ligand.
Sodium channel samples with and without added ethanol produced
identical CD spectra. Samples from several different preparations were
examined with the same results.
Circular Dichroism Analyses--
In the calculation of molar
ellipticity, a mean residue mass of 114.5 daltons was used. The
secondary structural analyses used DICHROWEB, an interactive webserver
(31) that permits analyses via the following methods: SELCON3 (32),
CONTIN (33, 34), CDSSTR (35), and K2D (36) with a wide range of protein
spectral databases (all derived from soluble proteins (37)). The
normalized root mean square deviation (NRMSD) parameter (38) was
calculated as a measure of the quality of the fit of the calculated
structure to the data. NRMSD values of <0.1 mean that the calculated
and experimental spectra are in close agreement (39).
Modeling of the Sodium Channel and Its Interaction with
LTG--
Multiple sequence alignment of the S6 transmembrane residues
from domains I, III, and IV was carried out using PSI-BLAST (40) and
displayed using Alscript (41) (Fig.
1).
Homology modeling of S5, the P-loops, and S6 from all four domains used
the open MthK channel x-ray structure (PDB accession number
1lnq) (42) as the framework, since LTG binds to the inactivated form of
the open channel. Areas of high homology between the sodium channel
domains and potassium channel were aligned, i.e. S5 and S6
transmembrane segments based on both homology and secondary structure
prediction. Non-homologous regions in the longer P-loops of domains I
and III, which correspond to putative glycosylation sites, were
deleted. The P-loops and N and C termini were modeled based on
homologous segments of the KcsA channel structure (PDB accession
number 1bl8).
Sodium channel sequences were aligned versus the MthK
channel using ClustalW (43), and the structure was modeled employing the program Modeler 4 (44). Rigid-body minimization and simulated annealing of the model was carried out using the program CNS (45).
Docking studies between ligand and the channel model used the
lamotrigine crystal structure (46) and the program AUTODOCK (47). The
regions of close association between the docked lamotrigine and the
sodium channel model were identified using the CCP4 (48) program CONTACT.
Purification of the Sodium Channel
The sodium channel from electric eel electroplax membranes was
purified using procedures similar to those previously described (23,
24) but with the following modifications: the anti-sialic acid horse
IgM affinity column was not eluted with colominic acid but instead was
eluted with a KCl gradient as this resulted in higher yields and less
degradation of the column, thus prolonging its useful lifetime. The
presence of an enhanced mixture of protease inhibitors and the use of
different buffers and detergent/lipid mixtures increased the yield and
stability of the preparation. The eluted sodium channel was detected as
a broad high molecular mass species on silver-stained gels (Fig.
2a) with an apparent molecular
mass of ~280-300 kDa and identified by Western blot analyses with
anti--helical conformation. The observed
changes are consistent with increased order involving the S6 segments
that form the pore, the domain III-IV linker, and the P-loops that
form the outer pore and selectivity filter. A molecular model has been
constructed for the sodium channel based on its homology with the
pore-forming regions of bacterial potassium channels, and automated
docking of the crystal structure of lamotrigine with this model
produces a structure in which the close contacts of the drug are with
the residues previously identified by mutational studies as forming the
binding site for this drug.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix. The C-terminal intracellular
domain has also been associated with a form of inactivation and a
postulated interaction with the N-terminal segment; its secondary
structure has been analyzed by circular dichroism
(CD)1 spectroscopy (12, 13).
The S4 transmembrane segments from domains I-IV have been identified
as the main voltage sensors (14), whereas the S5-S6 segments and the
intervening P-loop regions from domains I-V include residues
comprising the transmembrane pore-associated helices and the ion
selectivity filter (15).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-2,8 N-acetylneuraminic acid was extracted from
horse antiserum, a gift from Dr. R. Schneerson of the NICHD (22). Lamotrigine was a gift from Dr. R. W. Janes, Queen Mary College, University of London. Batrachotoxin was a gift from Dr. J. W. Daly of
the NIDDK. Polyacrylhydrazido-agarose, lipids, protease inhibitors, and
rabbit anti-horse IgG were purchased from Sigma. CNBr-activated
Sepharose 4B Fast Flow and PHAST gels were from Amersham
Biosciences and polyvinylidene fluoride membranes were from BDH Laboratory Supplies.
80 °C.
Membrane yield was ~20% of original electroplax tissue weight.
90 degrees) for
scattering samples such as membrane proteins (29). The instrument was
calibrated with camphor sulfonic acid for optical rotation and benzene
vapor for wavelength. Data were collected at 0.2-nm intervals, at a
constant temperature of 25 °C over a wavelength range from 300 to
185 nm. Five scans were collected for each protein sample and baseline
(consisting of the dialysate), and all samples were recorded in the
same 0.02-cm pathlength Suprasil cuvette. The averaged baseline
spectrum was subtracted from the averaged sample spectrum, and the net
spectrum smoothed with a Savitsky-Golay filter (30). Measurements were only made down to wavelengths where the instrument dynode voltage indicated that the detection was still within the linear range.
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Fig. 1.
Sequence alignment of residues of the
pore-forming S6 transmembrane segments of domains I, III, and IV, which
have been identified as the binding sites for pharmacological
agents. Sodium channel sequences from a broad cross-section of
species were aligned to illustrate sequence conservation in the pore
including: the electric eel E. electricus (P02719);
TTX-sensitive neuronal-expressed human sodium channels hNav1.1,
hNav1.2, hNav1.3, and hNav1.12; TTX-resistant human heart hNav1.5,
hNav1.8 expressed in neuroendocrine cells, and hNav1.11 expressed in
dorsal root ganglion neurons; tobacco budworm Heliothis
virescens (AF072493); the puffer fishes Fugu pardalis
(AB030482) and Fugu rubripes (D37977); sea squirt
Halocynthia roretzi (T43161); zebrafish Danio
rerio (AF297658); german cockroach Blattella germanica
(U73583); fruit fly Drosophila melanogaster (M32078);
Japanese firebelly newt Cynops pyrrhogaster
(AF123593); and California market squid Loligo opalescens
(T43167). Totally conserved residues are shown in black
relief, and highly conserved residues are in gray
relief. Residues shown by site-directed mutagenesis to be critical
for BTX binding are represented in blue, for lamotrigine
binding in red, and for both BTX and lamotrigine in
yellow.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-2,8-N-acetylneuraminic acid horse IgM (Fig. 2b). The electroplax sodium channel is reported to consist
of 30% by mass of carbohydrate, 12% sialic acid (9). Quantitative amino acid analyses were consistent with the reported sequence of the
electroplax sodium channel (data not shown). Negative stain electron
microscopy of the samples to be used for CD spectroscopy showed that
they contained micelles, each consisting of a single protein with
pseudo-4-fold symmetry and a central pore, as described earlier (16).
Dynamic light scattering studies also showed the CD samples to be
consistent with monomeric micellar
species.2
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Fig. 2.
Purification of sodium channels.
a, SDS-PAGE of affinity-purified sodium channels.
Lane A, molecular mass markers; lane B
solubilized membranes; lane C, flow through of affinity
column; lane D, purified sodium channels. b,
Western blots. lane A, solubilized membranes; lane
B, purified sodium channels.
Functional Characterization of the Sodium Channel
To demonstrate that the sodium channel retained functionality
after purification and reconstitution, single channel conductance experiments in planar lipid bilayers were carried in the presence of
BTX (Fig. 3a). BTX was used to
remove the fast inactivation process, thus allowing single-channel
events to be recorded with applied steady-state voltages. The
single-channel conductance of 17.5 pS from the I-V plot (Fig.
3b) is within the range of values published from similar
studies on the sodium channel (e.g. Ref. 49-51). The
identity and functionality of the channel under investigation was
confirmed by tetrodotoxin (TTX) block using 1 µM on both
sides.
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Structural Characterization of the Sodium Channel
CD Spectroscopy of Native Sodium Channels-- CD spectroscopy was used to examine the secondary structure of the purified sodium channel protein. CD spectra of membrane proteins in lipid bilayers are subject to distortions due to optical artifacts such as differential light scattering and absorption flattening (29). To avoid these effects, the CD spectra of the sodium channel were measured in lipid/detergent mixed micelles, where such effects are negligible (52).
The native sodium channel gives rise to a spectrum that is
characteristic of a protein with a high -helical content,
having peaks at ~221, 208, and
190 nm and a zero cross-over near
200 nm (Fig. 4a). Secondary
structural analyses were performed for all spectra using a range of
algorithms and reference databases derived from soluble proteins (31).
The analysis program CDSSTR (with reference database 3) consistently
produced the closest correspondence between experimental data sets and
calculated spectra (based on NRMSD values), so its results are
reported here: an
-helix content of 55% (Table I). However,
virtually identical results were found
for all methods tested. Indeed, nine different combinations of
algorithms and reference data bases produced solutions with n.r.m.s.d.
values of
0.1. The average value of the helix content from all these
was 56%; this high correspondence of results obtained by different
methods increases our confidence level in the veracity of the
calculated values.
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As a precaution, however, although the NRMSD values indicate a good correspondence between the calculated secondary structures and the actual structures, CD as a technique is most accurate in determining helix content. As a result, in this paper, discussions of the secondary structures will concentrate on the helix content of the protein. Furthermore, the interpretation of the analyses must bear in mind that all the reference data sets used in the analyses are derived from soluble proteins as they are the only ones currently available. Since the CD spectra of membrane proteins tend to have slightly different spectral characteristics (53), this could lead to some inaccuracy in the absolute values of the secondary structures calculated, although the changes in secondary structures measured will, in general, be unaffected.
Based on identification of helical transmembrane segments using
hydropathy analyses for transmembrane segments S1-S4 in all the
domains (~340 amino acids), analogy to the KcsA potassium channel
structure for the region from S5 to S6 (~300 amino acids) (54), and
analogy to the helical C-terminal domain of the human cardiac sodium
channel consisting of ~80 amino acids (12), the minimum -helical
content is estimated to be 720 residues, or 40% helical. The measured
value in this study is 55%, thus suggesting that the sodium channel
contains additional helical structures beyond those in the thus-far
identified helical regions.
CD Spectroscopy of Lamotrigine Binding to the Sodium Channel-- A large molar excess (120×) of LTG was added to the native sample to fully shift the equilibrium toward the conformation associated with the open inactivated state since in proteoliposomes the electrical membrane potential difference is zero. LTG itself does not give rise to a CD signal over the wavelength range examined. The spectral changes observed in the protein involved an increase in magnitudes of the peaks at 221 and 208 nm and a change in ratio between the 190 and 221 nm peaks. This type of spectral change is indicative of an increase in helix content, which was born out by the secondary structural calculations (Table I).
The increase in helical content was calculated to be ~9%, or as many as 160 amino acids. Thus binding to the helical regions of the S6 segments (19) appears to induce increased order in other parts of the structure, perhaps to the P-regions, which are partially helical, or in the III-IV loop region, which NMR studies on an isolated fragment (11) suggest has a propensity for helix formation. Large tertiary structural changes, but only small changes in secondary structure, have been detected between the crystal structures of open and closed bacterial potassium channels (42, 55). However, secondary structural changes involving the III-IV loop region, which induces fast inactivation and is thought to enter the inner mouth of the channel under these conditions would not have been detected in the KcsA channel, which only contains the S5-P-S6 regions. In addition, regulation in eukaryote sodium channels is expected to be somewhat different than that for the bacterial potassium channels since the sodium channels contain loop regions connecting homologous domains in the pseudotetramer instead of individual monomers, as in the homotetramer of the KscA channel.
To eliminate the possibility of changes resulting from nonspecific protein-drug interactions, CD spectra were collected for the flow-through fraction from electroplax membranes after affinity purification of the sodium channel, which contained other membrane proteins but no detectable sodium channel, with and without a similar amount of lamotrigine added. The spectra for the flow-through and the flow-through plus lamotrigine were identical (data not shown).
CD Spectroscopy of Batrachotoxin Binding to the Sodium Channel-- A 4-fold molar excess of BTX was used in the CD studies. The concentrations of BTX and sodium channel protein used were approximately the same as those used in the electrophysiology experiments and sufficient to cause steady-state activation of purified sodium channel reconstituted into planar lipid bilayers, i.e. the channels can be triggered to fluctuate between open and closed states under applied steady-state or continuous voltages.
As was found in the LTG experiments, upon BTX binding the CD spectrum
exhibited an increase in both the minima at 221 and 208 nm (Fig.
4b). The calculated secondary structure (Table I) indicated an increase (~6%) in -helix content similar to, but somewhat smaller than, the LTG effect. The differences between the LTG
and the BTX spectra are greater than the standard deviations of the
measurements, so the structures formed in the presence of LTG and BTX
are not identical. This is expected as they bind to different forms,
inactivated and activated, respectively, of the open channel.
Binding of both LTG and BTX--
It has been shown that BTX and
sodium channel-blocking drugs bind with adjacent overlapping but
non-identical sites in IVS6 (56), IIIS6 (19, 57), IIS56 (58), and IS6
(59) transmembrane segments (Fig. 1). In addition, higher
concentrations of local anesthetics can block sodium channels activated
by BTX without displacing BTX (60). CD data were collected for the
native channel in the presence of both LTG and BTX (Fig.
4b). The spectral changes observed in the 221 and 208 peaks
upon binding both the toxin and the drug were larger than for either
alone, and there is an apparent 13% increase in -helical
conformation (larger than either alone) (Table I). Thus it appears that
there may be a further augmentation and stabilization of helical
structure on binding of both BTX and LTG to the S6 segments of the
inner pore of the sodium channel.
Model of the Sodium Channel Pore and Its Interactions with LTG and BTX
A molecular model that could be used to investigate the
molecular nature of the binding of LTG in the pore was created by aligning residues 19-98, i.e. the pore-forming residues of
the x-ray structure of the open form of a bacterial potassium channel, with residues 238-408 of domain I, residues 691-797 of domain II,
residues 1127-1270 of domain III, and residues 1447-1547 of domain IV
of the sodium channel. Residues 23-119 of the KcsA potassium channel
x-ray structure were also used to model the P-loop regions and the N-
and C-terminal residues of these segments. The orientations of the four
domains were modeled by aligning domains I-IV of the sodium channel
with chains A-D of the MthK structure in a clockwise arrangement as viewed from the extracellular surface (61). Residues forming the binding sites for LTG (as identified by mutational studies)
all appear to lie on the pore-facing side of the S6 helices from
domains I, III, and IV (Fig. 5).
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The crystal coordinates for lamotrigine (46) were docked into this
model using the program AUTODOCK. The highest ranking docked position
has a final energy of 4.29 kcal/mol and on analysis gave consistent
results with respect to the mutation studies. In the docked position,
residues A1252 and L1256 of IIIS6, identified as the putative binding
site lamotrigine by the mutation studies, make van der Waals contacts
mainly with the triazine ring structure of lamotrigine and also one of
the chlorine atoms of the dichlorophenyl ring structure, while F1555
and V1559 of IVS6, also identified by mutation studies as contributing
to a lamotrigine binding site, make contacts chiefly with the
dichlorophenyl ring.
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DISCUSSION |
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Many drugs including local anesthetic compounds and anticonvulsant
drugs such as lamotrigine block sodium channels in a
voltage-dependent manner (19). When membranes are
hyperpolarized block is minimal, but depolarization induces significant
block. Lamotrigine blocks the channel (Fig.
6) in a
use/frequency-dependent manner (62) and was found to block
sustained repetitive firing of sodium-dependent action
potentials in mouse spinal cord cultured neurons (63) and hence is used
in the treatment of seizures. It has been proposed that use dependence
arises from preferential binding to the inactivation states, which
develop in response to rapid repetitive opening (62). However, although
lamotrigine may bind with different affinities in a
concentration-dependent manner to several discrete dynamic
states of the sodium channel, in this study only overall binding was
explored in the absence of any applied membrane potential. Wild-type
rat brain sodium channels have been shown to be inhibited by
lamotrigine with a dissociation constant ranging from 12 to 32 µM (62, 64), i.e. in the same range as is
required for the treatment of epilepsy in humans (63). The 50 µM used in these studies mimics these pharmacological and
therapeutic concentrations.
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Batrachotoxin is a lipid-soluble alkaloid that also binds specifically to sodium channels in a state-dependent manner (21) (Fig. 6). The main effects of BTX binding are a shift in the voltage dependence of activation in a hyperpolarizing direction, allowing steady-state activation of the channel, the inhibition of both fast and slow inactivation, and a small increased single-channel conductance coupled with a slightly altered ion selectivity (49). BTX in the micromolar concentration range causes steady-state activation of purified sodium channels reconstituted in phospholipid vesicles. A number of previous studies reported the electrical properties of BTX-modified E. electricus sodium channels in planar lipid bilayers. Single channel conductances of 15-25 pS were measured in various planar lipid bilayer systems (electrolyte: 0.5 M NaCl) (49-51). In the current study, a single-channel conductance of ~17-18 pS was measured in solvent-free, negatively charged lipid bilayers formed at a patch pipette tip (Fig. 3a). BTX was incorporated into vesicles containing sodium channels in the absence of a membrane potential. For the CD studies in mixed micelles, the sodium channels are not subjected to any membrane potential; therefore it was anticipated that BTX binding would stabilize the open conformation or one of the conformational transitions accompanying formation of the activated state.
In this study we examined conformational changes associated with
formation of open states of the channel and found that the helix
content increased dramatically. These conformational changes could
involve the S6 segments, the P-regions, which form the outer pore and
selectivity filter (although not exclusively as a recent study suggests
(65)), or the III-IV cytoplasmic linker and the C terminus, which are
involved in inactivation processes. BTX and LTG have been shown to have
distinct and overlapping molecular determinants for binding in the
inner pore. The common sites for state-dependent binding
are residues on the same face of the IS6, IIIS6, and IVS6 helices (Fig.
5) (19). The structure of the KcsA channel (54) has established an
-helical architecture for the inner pore, which is assumed to be
present in all members of the family of homologous ion channels (66)
including by analogy the S6 segments of the sodium channel. These S6
transmembrane segments are thought to rotate during channel activation
leading to local changes in side-chain interactions with contiguous
transmembrane helices (67). Of the three S6 segments involved in
binding, IIIS6 appears to be the most highly conserved and IVS6 shows
the highest sequence variation (Fig. 1). However, in all three S6 segments, the residues identified as forming the drug binding site are
all highly conserved, predominantly hydrophobic, and demonstrate a
marked periodicity of every three to four residues across a large
spectrum of species. Furthermore, mutations to alanine of two residues,
Leu-1465 and Ile-1469 of the rat brain IIA sodium channel
(corresponding to Leu-17 and Ile-21 of IIS6 in Fig. 1) both decreased
lamotrigine binding and caused a positive shift in the steady-state
activation curve (19). These residues, which contribute to stabilizing
the open state, are all disposed along one face of the inner pore helix
and are proposed to face the lumen of the pore in the activated and
inactivated open states of the channel (Fig. 5).
CD analyses of the KcsA channel performed over a range of different pH values to induce a conformational change (67) indicated that there was no significant change in secondary structure. This suggested that pH-dependent channel opening must be coupled to the motion of entire domains or secondary structure elements in this truncated channel. An analysis of the voltage-gated potassium channel using cysteine modifications of the S6 segment led to the conclusion that the presence of a Pro-X-Pro motif in these helices would give rise to a `bent S6' helix model as a variation on the KcsA model (68). The voltage-gated sodium channel S6 segments do not have this motif, but the presence of conserved glycines in three of four of these helices can lead to the proposal that the inner and outer parts of S6 segments could move independently during activation gating. However, rigid body movements of S6 segments are unlikely to be the source of the observed spectral changes, which clearly result from secondary structure conformational changes.
Other possible sources for the observed conformational change are the P-loops or the III-IV linker responsible for inactivation. There is evidence for conformational flexibility in the P-loops. Indeed, investigation of the Drk1 K channel identified subconductance states relating to incomplete channel opening due to incomplete pore formation (69). Mutant Shaker K+ channels allowed recording of these subconductance states suggesting the existence of different conformations of the selectivity filter reflecting incomplete pore formation giving rise to distinct conductance rates. In contrast to this result, spin labeling experiments on the non-voltage-gated KcsA K+ channel led to the conclusion that the selectivity filter remained immobile during gating (67). Finally, mutagenesis data has implicated the S4-S5 intracellular linker of domain IV in the sodium channel as the probable receptor for the inactivation particle (70). In the current work, the simultaneous addition of LTG and BTX suggests binding to the open inactivated state so the observed secondary structure change could involve conformational changes associated with inactivation particle binding.
On the bases of the CD spectral changes observed in the present study,
we propose a model for activation and inactivation that involves a
significant secondary structural conformation change in addition to the
rigid body movements of the S6 transmembrane helices previously
inferred. This is likely to involve the inner pore helices, P regions,
and the III-IV interdomain linker.
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ACKNOWLEDGEMENTS |
---|
We thank Peter Sherritt of the Protein and Nucleic Acid Chemistry Facility at the University of Cambridge for the quantitative amino acid analyses, Dr. Robert W. Janes of Queen Mary College, University of London for lamotrigine, Dr. R. Schneerson of the National Institutes of Health for the horse antiserum, Dr. J. W. Daly of the National Institutes of Health for batrachotoxin, and Dr. Ulrich Gohlke, Birkbeck College, University of London for help with the electron microscopy. H. D. acknowledges advice and comments by Dr. S. Bendahhou (Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Sophia-Antipolis, France) and thanks Dr. J. Dempster of Strathclyde University (Glasgow, UK) for free electrophysiology acquisition and analysis software.
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FOOTNOTES |
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* This work was funded by Project Grant B15499 from the Biotechnology and Biological Sciences Research Council (BBSRC) (to B. A. W.), a joint travel grant from the Royal Society and the CNRS (to B. A. W. and H. D.), and an equipment grant from the CNRS (to H. D.). The circular dichroism instrumentation was supported, in part, by Grant B14225 from the BBSRC (to B. A. W.).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.
§ Supported by the British Heart Foundation H. W. Fletcher Ph.D. Studentship.
To whom correspondence should be addressed. Tel.:
44-207-631-6857; Fax: 44-207-631-6803; E-mail:
ubcg25a@mail.cryst.bbk.ac.uk.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M208356200
2 N. B. Cronin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CD, circular dichroism; LTG, lamotrigine; BTX, batrachotoxin; NRMSD, normalized root mean square deviation; TTX, tetrodotoxin; pS, picosiemens.
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