(Received for publication, December 26, 1995)
From the
The binding site for batrachotoxin, a lipid-soluble neurotoxin
acting at Na channel receptor site 2, was localized
using a photoreactive, radiolabeled batrachotoxin derivative to
covalently label purified and reconstituted rat brain Na
channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive
allosteric enhancers of batrachotoxin binding, a protein with an
apparent molecular mass of 240 kDa corresponding to the
Na
channel
subunit was specifically covalently
labeled. The region of the
subunit specifically photolabeled by
the photoreactive batrachotoxin derivative was identified by antibody
mapping of proteolytic fragments. Even after extensive trypsinization,
an anti-peptide antibody recognizing an amino acid sequence adjacent to
Na
channel transmembrane segment IS6 was able to
immunoprecipitate up to 70% of the labeled peptides. Analysis of a more
complete digestion with trypsin or V8 protease indicated that the
batrachotoxin receptor site is formed in part by a portion of domain I.
The identification of a specifically immunoprecipitated photolabeled
7.3-kDa peptide containing transmembrane segment S6 from domain I
restricted the site of labeling to residues Asn-388 to Glu-429 if V8
protease digestion was complete or Leu-380 to Glu-429 if digestion was
incomplete. These results implicate the S6 transmembrane region of
domain I of the Na
channel
subunit as an
important component of the batrachotoxin receptor site.
Batrachotoxin (BTX) ()is a steroidal alkaloid toxin
from skin secretions of South American frogs, Phyllobates
aurotaenia and Phyllobates terribilus, which are used by
the native Indians of Colombia to make poison blowdarts and
arrows(1) . It is one of the most toxic nonproteinaceous
substances known and is capable of inducing membrane depolarization at
concentrations in the low nanomolar range. The voltage-gated
Na
channels of excitable membranes are the molecular
targets of BTX, and all aspects of Na
channel function
are altered upon exposure to BTX: inactivation is blocked, single
channel conductance is decreased, voltage dependence of activation is
shifted to more negative potentials, and selectivity for Na
is impaired (reviewed in (2) and (3) ).
Competitive binding studies with radiolabeled neurotoxin analogues
have distinguished five distinct receptor sites for neurotoxins on the
Na channel, including neurotoxin receptor site 2 which
is occupied by the full agonist BTX and the partial agonists
veratridine, aconitine, and grayanotoxin which modulate Na
channel gating(2, 3) . The binding of BTX at
neurotoxin receptor site 2 results in stabilization of an open
conformation of the channel. At least four of the five receptor sites
have been shown to be located on the 260-kDa
subunit, which is
composed of four homologous domains (I-IV) containing six
putative transmembrane segments (S1-S6; Refs. 4, 5).
Site-directed mutagenesis experiments have identified amino acid
residues of the
subunit that are required for high affinity
binding of tetrodotoxin and saxitoxin at neurotoxin receptor site
1(6, 7, 8) . Peptide segments from neurotoxin
receptor site 3, which binds
-scorpion toxins and sea anemone
toxins, and neurotoxin receptor site 5, which binds brevetoxins, have
been identified by photoaffinity labeling and peptide mapping with
sequence-specific antibodies(9, 10, 11) , and
individual amino acid residues that are required for high affinity
binding of
-scorpion toxins and sea anemone toxins to receptor
site 3 have been identified by site-directed mutagenesis(12) .
BTX binding to neurotoxin receptor site 2 is allosterically
modulated by other Na channel neurotoxins. The
-polypeptide toxins from scorpion and sea anemone(13) ,
the
-cyano-pyrethroid
insecticides(3, 14, 15) , and brevetoxin (14, 15) all enhance BTX binding. Other compounds such
as tetrodotoxin and saxitoxin(16) , local
anesthetics(17, 18) , and the anticonvulsants
diphenylhydantoin and carbamazepine (19) decrease BTX-binding
affinity. These results indicate that the BTX-binding site is
particularly sensitive to conformational alterations induced at
distinct sites.
Neurotoxin receptor site 2 is present on
Na channels expressed from
subunit cDNA
alone(20, 21) . Mapping of the peptide segments of the
subunit that form receptor site 2 by photoaffinity labeling has
not been feasible because the ligands binding at that site have
relatively low affinity and are hydrophobic, resulting in low specific
binding values. These difficulties can be circumvented by taking
advantage of the allosteric enhancement of BTX binding by pyrethroids
and the brevetoxin analogue, PbTx-1. This combination of effectors can
enhance BTX binding in a purified and reconstituted Na
channel preparation up to 1000-fold, reducing the K
to a value below 1 nM(15) . In this study, this combination of toxins is used
to enhance high affinity binding and covalent incorporation of a
photoreactive, radiolabeled BTX derivative,
[
H]BTX-OAB, into purified Na
channels, and the major site of incorporation is identified by
peptide mapping with anti-peptide antibodies.
Figure 1:
Structure of BTX
and its derivatives. The positions of H labels are shown by asterisks. Batrachotoxinin-A (BTX-A) is the
nontoxic natural precursor of batrachotoxin from which both
radiolabeled derivatives (bottom two structures) were
synthesized.
Figure 2:
Competitive displacement of
[H]BTX-B bound to reconstituted Na
channels by unlabeled veratridine. Binding of
[
H]BTX-B (17 nM, solid circles)
and [
H]BTX-OAB (10 nM, solid
squares) to reconstituted Na
channels was
measured as described under ``Experimental Procedures'' in
the presence of the indicated concentrations of veratridine. Control
binding of [
H]BTX-B to heat-inactivated and
reconstituted Na
channels (open circles) and
reconstituted phospholipids (open squares) was also
determined.
Figure 3:
Enhancement of specific
[H]BTX-B binding by RU39568, RU51049, and PbTx-1. A, [
H]BTX-B binding to reconstituted
Na
channels was measured as described under
``Experimental Procedures'' in the presence of 5 nM [
H]BTX-B and increasing concentrations of
RU39568 (solid circles) or RU51049 (solid squares). B, [
H]BTX-B binding was measured in the
presence of 0.5 nM [
H]BTX-B and 10
µM RU pyrethroid with and without 100 nM PbTx-1
in the presence (stippled bars) or absence (solid
bars) of 300 µM veratridine for determination of
nonspecific and total binding, respectively. Binding of
[
H]BTX-B to purified soluble Na
channel was measured in the presence of 10 µM RU51049 and 100 nM PbTx-1. Samples were incubated at 25
°C for 17 h for determination of maximum allosteric
interaction.
Figure 4:
Specific photolabeling of purified
Na channels. Reconstituted Na
channels were purified and photolabeled with 90 nM [
H]BTX-OAB as described under
``Experimental Procedures'' in the absence (solid
circles) or presence (open circles) of 300 µM veratridine. Samples were analyzed by SDS-PAGE (7% porous reducing
gel system; Doucet et al.(32) ), and the incorporation
of photolabel was determined in 3-mm gel slices by extraction and
scintillation counting as described under ``Experimental
Procedures.'' The migration of molecular mass standards (kDa) are
indicated by arrows.
Figure 5:
Immunoprecipitation of peptides covalently
labeled by [H]BTX-OAB. A, recognition
sites of anti-peptide antibodies. Antibodies directed against synthetic
peptides corresponding to different sequences of the
subunit of
the type IIA sodium channel were prepared as described (Gordon et
al.(25, 26) ). Antibodies were directed against
synthetic sodium channel peptides corresponding to the amino acid
sequences 355-372 (SP31), 382-400 (SP28),
427-445 (SP1), 468-504 (SP11),
531-547 (SP32), 708-722 (SP15),
1145-1164 (SP20), 1480-1498 (SP14),
1541-1561 (SP19), 1736-1753 (SP29),
1789-1798 (SP13). B, immunoprecipitation of
[
H]BTX-OAB-labeled Na
channel
peptide fragments from proteolytic cleavage by trypsin. Photolabeled
reconstituted Na
channels were digested with
increasing concentrations of TPCK-trypsin, and the resulting peptide
fragments were probed with the indicated antibodies as described under
``Experimental Procedures.'' TPCK-trypsin concentrations for
each antibody treatment were as follows (from left to right): 0.3, 1,
10, 100 µg/ml. Values are expressed as the percentage of total cpm
immunoprecipitated without trypsin
treatment.
The site of BTX covalent labeling of the
Na channel
subunit was more precisely localized
by more extensive proteolytic cleavage at lysine and arginine residues
with trypsin and at glutamic acid and aspartic acid residues with V8
protease from S. aureus. Under the conditions for proteolysis
described in Fig. 6A, approximately 50% of the total
radioactivity was precipitable by anti-SP11 antibody and 70% by
anti-SP1 antibody after cleavage with 10 µg/ml trypsin for 1 h.
These antibodies, directed against peptides within the first 60 amino
acids at the intracellular side of transmembrane segment IS6,
completely lose their ability to recognize
subunit-bound
[
H]BTX-OAB upon digestion with 100 µg/ml V8
protease for 1 h (Fig. 6B). However, anti-SP31 and
anti-SP28, antibodies directed against peptides within the first 50
amino acids extracellular to transmembrane segment IVS6, were able to
precipitate 30-60% of radioactivity after cleavage with trypsin (Fig. 6A) and over 50% of the total specific
radioactivity after cleavage with V8 protease (Fig. 6B). Anti-SP15, anti-SP14, and anti-SP19, which
are directed against sequences farther toward the carboxyl terminus, do
not immunoprecipitate a significant amount of photolabel (Fig. 6, A and B). These results indicate that
the site of [
H]BTX-OAB covalent labeling is
located to the amino-terminal side of the anti-SP1 recognition peptide.
Figure 6:
Immunoprecipitation of peptides labeled by
[H]BTX-OAB after proteolytic cleavage by
TPCK-trypsin and V8 protease. Photolabeled and reconstituted
Na
channel was digested in the presence of 10
µg/ml TPCK-trypsin (A) or 100 µg/ml V8 protease (B) for 2 h at 37 °C, and the resulting peptide fragments
were probed with the indicated antibodies as described under
``Experimental Procedures.'' Values are expressed as the
percentage of total cpm immunoprecipitated by each antibody with no
prior protease treatment.
Figure 7:
SDS-PAGE analysis of tryptic peptides from
[H]BTX-OAB-labeled Na
channel. A, samples were labeled with [
H]BTX-OAB,
digested with 100 µg/ml TPCK-trypsin at 37 °C overnight, then
with 100 µg/ml V8 protease at 37 °C overnight, and analyzed on
a Tricine SDS-PAGE gel (system 2) as described under
``Experimental Procedures.'' The labeled peptides
precipitated by anti-SP28 (solid triangles), anti-SP1 (solid circles), or preimmune rabbit IgG (open
circles) are shown. The migration positions of molecular mass
standards are indicated by the arrows. The gel dye front is
indicated by df. B, estimations of molecular size of
the [
H]BTX-OAB-labeled peptide are shown. The
three different gel systems used are described under
``Experimental Procedures.'' The average molecular size
determined in these four experiments is 7.3
kDa.
Experiments like the one illustrated in Fig. 7were carried out with several preparations of purified and
reconstituted Na channels, two different preparations
of [
H]BTX-OAB, and three different SDS-PAGE
separation systems with similar results. Although the small
6-7-kDa peptide, which can be precipitated by anti-SP28, migrates
close to the dye front in all of the SDS-PAGE systems used, the
estimation of size is consistent in all three gel systems (Fig. 7B). These results indicate that
[
H]BTX-OAB is covalently incorporated into a
peptide of about 7.3 kDa containing the anti-SP28 antibody recognition
site.
Examination of the amino acid sequence of the subunit
near the SP28 peptide (Fig. 8) allows identification of the
photolabeled fragment obtained from extensive cleavage with trypsin and
V8 protease. In domain I, the peptide which includes all of the
anti-SP28 recognition sequence from the trypsin cleavage site, Leu-380,
to the V8 protease cleavage site, Glu-429, has a calculated molecular
mass of 7.3 kDa (Fig. 8). Trypsin cleavage within peptide SP28
was previously found to occur only under extreme
conditions(9) , so cleavage at Arg-395 or Lys-399 is unlikely.
More complete cleavage by V8 protease at Glu-387 within the SP28
sequence might yield the peptide from Asn-388 to Glu-429 with a
calculated molecular mass of 6.0 kDa, which contains all but 6 residues
of the sequence of peptide SP28. Because peptides are linked through
their amino termini to bovine serum albumin for immunization, the first
few amino acid residues of the peptide sequence may not be essential
for antibody recognition of the peptide. Thus, the possible
[
H]BTX-OAB-binding peptides containing the SP28
sequence begin 13 or 21 amino acid residues on the extracellular side
of transmembrane segment IVS6, contain the entire transmembrane segment
IVS6, and 3 amino acids on the intracellular side of segment IVS6.
Figure 8:
Amino
acid sequence of the batrachotoxin-binding region on the Na channel
subunit. The primary structure and antibody
recognition sequences of the photolabeled peptides from domain I are
shown. Antibody recognition sequences (underlined),
transmembrane segments (boxed), and expected sites of trypsin (arrows) and V8 protease (asterisks) cleavage are
indicated.
Less extensive cleavage with trypsin alone yields peptides that
contain the recognition sites for anti-SP1 and anti-SP11 on the
intracellular, carboxyl-terminal side of transmembrane segment IS6 (Fig. 6A). These peptides must contain sequences
extending beyond Ser-504 (Fig. 8). In contrast, cleavage with V8
protease removes the recognition site for these two antibodies (Fig. 6B) by cleavage at one of the numerous Glu
residues in the SP1 sequence (Fig. 8). Cleavage of the SP1
peptide from transmembrane segment IS6 by treatment with V8 protease
and resistance of the SP28 peptide within the subunit to cleavage
by trypsin as observed here are consistent with previous work on
peptide mapping of the
-scorpion toxin receptor
site(9, 34) .
We have found that in the
presence of the pyrethroid, RU51049, and the brevetoxin, PbTx-1, both
[H]BTX-B and [
H]BTX-OAB
bound to purified and reconstituted sodium channels with similar high
affinity. No high affinity binding of [
H]BTX-B
was present in heat-inactivated sodium channel preparations or to
phospholipid alone. These results establish that high affinity binding
of BTX derivatives requires the native conformation of the purified
sodium channel and that neurotoxin receptor site 2 is present in active
form on the solubilized, purified, and reconstituted sodium channel.
Covalent labeling of purified and reconstituted Na
channel with [
H]BTX-OAB provided the
initial material used for biochemical localization of a peptide
sequence that contributes to formation of neurotoxin receptor site 2.
Further localization of the site of covalent labeling
by [H]BTX-OAB was achieved by analysis of
immunoprecipitated fragments with antibodies recognizing peptide
sequences within or near domain I after more extensive proteolytic
cleavage with TPCK-trypsin and V8 protease. After trypsinization, two
anti-peptide antibodies recognizing the amino acid sequence on the
intracellular side of transmembrane segment IS6 were able to
immunoprecipitate at least 50% of the
[
H]BTX-OAB-labeled peptides, and two anti-peptide
antibodies recognizing the amino acid sequence on the extracellular
side of this same transmembrane region were able to immunoprecipitate
30-60% of the labeled peptides. In contrast, after treatment with
V8 protease, the ability of the two antibodies recognizing the
intracellular amino acid sequences to precipitate the labeled peptide
was completely lost, whereas the antibodies recognizing the sequences
extracellular to IS6 retained their ability to precipitate the labeled
peptide. These results indicate that the BTX receptor site is formed by
a portion of domain I and that the site of photolabeling does not
include the amino acids to the carboxyl-terminal side of transmembrane
region IS6.
A more precise biochemical localization of the site of
covalent labeling by [H]BTX-OAB was achieved by
SDS-PAGE analysis of cleavage products from proteolytic digestions of
labeled Na
channels with both TPCK-trypsin and V8
protease. The identification of a specifically immunoprecipitated 7-kDa
peptide from domain I restricts the labeled peptide fragment to
residues Asn-388 to Glu-429 (calculated molecular mass, 6.0 kDa, Fig. 8) or to the region from Leu-380 to Glu-429 (7.3 kDa) if V8
protease cleavage is incomplete. When estimations of molecular mass
from three different gel systems are averaged together, the value for
the radiolabeled, immunoprecipitated peptide is 7.3 kDa, in excellent
agreement with the size of the predicted cleavage product from Leu-380
to Glu-429.
The photoreactive azide of
[H]BTX-OAB is positioned on an aromatic side
chain known to be important in conferring toxicity to BTX because the
precursor BTX-A, which lacks this aromatic moiety, is
nontoxic(3) . Therefore, the photoreactive side chain most
likely interacts with an integral part of the receptor site for BTX on
the Na
channel
subunit. Thus, our results
implicate transmembrane segment IS6 in direct interaction with bound
BTX in receptor site 2.
BTX binding is also affected by binding of
toxins to more distant regions of the Na channel.
Site-directed mutagenesis studies have identified the amino acid
residues lining the mouth of the channel pore between transmembrane
segments 5 and 6 in each of the four domains as critical for binding of
tetrodotoxin and saxitoxin at neurotoxin receptor site
1(6, 7, 8) . The binding of these toxins to
site 1 has been shown to allosterically decrease the binding of BTX to
site 2 under some conditions(16) .
The interactive nature of
BTX binding provides evidence that neurotoxin receptor site 2 is
located in a region of the Na channel that exhibits
considerable structural flexibility. Not only does the binding of other
toxins affect the affinity of BTX binding through allosteric alteration
of site 2, but binding of BTX itself results in a conformational change
of the channel. Upon BTX binding to the Na
channel,
the flux of relatively impermeant cations increases, indicating that
the size of the ion selectivity filter is increased, inactivation is
blocked, and the voltage dependence of activation is shifted to more
negative potentials (see review by Brown et al.(42) ).
These alterations in channel function indicate that the Na
channel has multiple neurotoxin binding sites that are located in
areas of great conformational flexibility. Upon binding of neurotoxins
to the Na
channel, conformational changes occur that
alter the position of toxin binding sites with respect to one another
and change channel gating kinetics. By locating the precise positions
of toxin receptor sites and by understanding the relationship of these
sites to one another, the alterations of normal gating processes
following toxin binding and mechanisms of allosteric interaction among
bound toxins will be clarified.