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(Received for publication, March 23, 1995; and in revised form, June 30, 1995) From the
Epitopes for monoclonal antibodies directed against the purified
adult rat skeletal muscle sodium channel (rSkM1) were localized using
channel proteolysis and fusion proteins. The interactions between these
and other monoclonal antibodies with site-specific polyclonal
antibodies were used to investigate the spatial relationships among
rSkM1 cytoplasmic segments. Competition between antibodies for binding
was performed using a solution-phase assay in which solubilized channel
protein retains many of the biophysical characteristics of the rSkM1
protein in vivo. Our results support a model in which: 1) the
amino terminus assumes a rigid structure having a fixed orientation
with respect to other intracellular segments; 2) the interdomain
2-3 region is centrally located on the cytoplasmic surface of the
channel, extends farther into the cytoplasm, and has an intermediate
degree of flexibility; 3) the beginning of the amino terminus and end
of the carboxyl terminus specifically interact with each other; and 4)
domains 1 and 4 are adjacent. The sequences responsible for the
interaction of the amino and carboxyl termini were identified by
demonstrating the specific binding of a synthetic peptide encompassing
the first 30 residues of the rSkM1 amino terminus to a fusion protein
containing the rSkM1 carboxyl terminus.
In studies of voltage-dependent ion channels, a major goal is to
correlate specific aspects of protein structure with channel function.
In order to attain this goal, an accurate model of channel tertiary
structure is required. Sequence information for a variety of
voltage-dependent sodium channels is now available, providing the basis
for several models of channel tertiary structure (1, 2, 3, 4, 5) . All
current models postulate the presence of four membrane-embedded
homologous domains joined by cytoplasmic linking and terminal
sequences. Although the models were initially based largely on
theoretical considerations, various aspects have been tested using a
variety of molecular, biochemical, and immunological techniques, and
their general features have been validated. In previous studies, we
used a combination of limited proteolysis and antibody binding to
provide experimental support for the presence of four compact repeat
domains in the skeletal muscle sodium channel, to identify the
topography of the regions that join and flank these domains, and to
probe the relative orientation of the large extramembrane cytoplasmic
elements(6, 7, 8, 9, 10, 11) .
This work also allowed us to map the location of epitopes for
monoclonal antibodies we had previously generated against purified
sodium channel protein. Our binding studies divided these antibodies
into two large groups based on mutually exclusive
competition(11) . While epitopes in each group were typically
clustered in similar regions of the channel sequence, in several cases
we found monoclonals in the same group that recognized epitopes widely
separated in the primary sequence but presumably brought together in
the native protein by folding of the polypeptide
backbone(6, 12) . Identification of similar
interactions that reflect tertiary rather than primary structure can
provide useful information about the structural organization of the
channel's cytoplasmic domains and form the basis for the work
reported here. In this study, we first localize epitopes for
additional monoclonals against the channel. Competition between our
monoclonal panel and polyclonal antibodies developed against defined
channel oligopeptides is then used to probe the spatial organization of
epitopes in the native channel. Using this approach, we have developed
a model for the organization of the channel cytoplasmic domains and
have identified a specific interaction between the channel's
amino and carboxyl termini.
Figure 1:
Antibody location and relative
position. Two-dimensional model of the sodium channel with the relative
positions of each antibody used in this study. Monoclonal antibodies
are indicated by boxes, polyclonal antibodies by ovals. The amino terminus is present on the left side while the carboxyl terminus is present on the right side of the figure. D1 refers to domain 1, D2 to
domain 2, etc. Refer to Table 1for details of the residues
comprising each antibody epitope.
Figure 2:
Initial localization of F/C11 and B/D6
epitopes. Western blot depicting nonproteolyzed (A) and limit
digests (B) of rSkM1 protein developed with the indicated
antibody. Limit digests were obtained by treating 5 pmol of purified
sodium channel protein for 120 min with
1-chloro-3-tosylamido-7-amino-2-hepanone-
These epitopes were further
localized to the interdomain 2-3 region on the basis of antibody
binding to a fusion protein that contained residues 794-1014 in the
rSkM1 sequence. Both antibodies reacted specifically with this fusion
protein both on Western blots and in radioimmunoassay. We refined this
localization using fusion proteins containing successively smaller
fragments of the interdomain 2-3 region. These fusion proteins
were constructed using either naturally occurring restriction sites in
the coding sequence or synthetic primers in conjunction with the
polymerase chain reaction (see Sun et al.(14) for
details). Our binding data (Fig. 3) restricts the epitope for
F/C11 to residues 865-875 (mid-portion of interdomain 2-3)
and the epitope for B/D6 to residues 965-975 (carboxyl-terminal
half of interdomain 2-3 just prior to domain 3) in the rSkM1
sequence.
Figure 3:
Localization of monoclonal epitopes using
fusion proteins. Schematic diagram of the panel of fusion proteins used
to localize the epitopes for F/C11 and B/D6. AA refers to
amino acid residues in the rSkM1 protein sequence. A
``+'' indicates that the respective antibody identified
the indicated fusion protein in radioimmunoassays and on Western blots,
while a ``-'' indicates that there was no reactivity of
the indicated fusion protein with the respective
antibody.
Polyclonal antibodies directed against epitopes
in the amino and carboxyl termini and each of the three interdomain
regions were then examined individually for interactions with the four
representative monoclonal antibodies. The binding of amino-terminal
monoclonal antibodies A/B2 and L/D3 (residues 1-6 and
19-24, respectively) (12) was not affected by prior
equilibration of the channel with polyclonal antibody I-31 (residues
31-46) (Fig. 4A). This result was unexpected,
since the epitopes for all three antibodies lie within the first 46
amino acids of the rSkM1 amino terminus. A/B2 and L/D3 were then
separately screened for competition with polyclonal antibodies directed
against specific regions in the interdomain 1-2 (I-467),
interdomain 2-3 (B-30), interdomain 3-4 (R-12), and
carboxyl terminus (B-23). A/B2 did not interact with any of these
site-directed polyclonal antibodies. For L/D3, however, competition was
observed with both B-30 (interdomain 2-3) and B-23 (proximal
carboxyl terminus) (Table 2). The amount of monoclonal antibody
bound at 4 h of incubation declined to approximately 35% of control as
the concentration of polyclonal antiserum used for preincubation
increased from 1:100,000 to 1:1.
Figure 4:
Three patterns of antibody competition
observed in this study. Polyclonal antibody was first incubated with
immobilized sodium channel protein in a solution phase assay at the
indicated dilutions of cell culture supernatants. After washing, one of
four monoclonal antibodies was incubated with immobilized sodium
channel and bound monoclonal antibody quantitated by the binding of
iodinated goat anti-mouse IgG. All experiments were repeated twice with
three samples per experimental point. Data are reported as the average
of three samples with error bars indicating standard
deviation. A, amino-terminal polyclonal antibody I-31
sterically hinders the binding of monoclonal antibodies F/C11 and B/D6
to the interdomain 2-3 region (approximately 900 residues distant
from the amino terminus) but has no effect on the binding of two
monoclonal antibodies (A/B2 and L/D3) whose epitopes
are located immediately adjacent in the amino terminus of the channel.
These data support a model in which the amino terminus is in a compact,
folded, rigid structure and the 2-3 interdomain region is
centrally located. B, binding of I-1771 inhibits subsequent
A/B2 and L/D3 binding with a steep concentration dependence, suggesting
that the near amino- and distal carboxyl termini of the sodium channel
are located less than 3.5 nm apart in the native
channel.
When monoclonal antibodies against
epitopes in the interdomain 2-3 region (F/C11 and B/D6) were
examined for competition with the same panel of polyclonal antibodies,
a second pattern of interactions was observed. In this case, each of
the polyclonal antibodies interfered with the binding of both F/C11 and
B/D6 (Fig. 4A and Table 2). For each of these
monoclonal-polyclonal antibody pairs, competition was incomplete and
again increased gradually over a wide range of antibody concentrations,
suggesting that antibodies bound to the two epitopes might occupy
partially overlapping volumes but that neither antibody completely
blocked access to the other epitope. A third pattern of competition
was consistently observed when polyclonal antibody I-1771 (directed
against residues 1771-1791 at the distal end of the carboxyl
terminus) was assessed with respect to monoclonal antibodies directed
against epitopes located at or near the beginning of the amino
terminus. I-1771 inhibited the binding of amino-terminal monoclonal
antibodies A/B2 and L/D3 with a steep concentration dependence,
decreasing monoclonal antibody binding by 80% over a single log unit of
polyclonal antiserum dilution (Fig. 4B). This pattern
of competition suggests mutually exclusive antibody binding to these
two epitopes. This does not represent antibody cross-reactivity between
the amino- and carboxyl-terminal epitopes since we have previously
shown that each antibody recognizes only channel fragments containing
its cognate epitope(8) . A more plausible explanation is that
the amino and carboxyl termini lie in close proximity and, perhaps,
physically interact. We tested this hypothesis by examining the
interactions between fragments of various channel cytoplasmic segments
in a solution phase binding assay. Fusion proteins containing the
bacterial MBP joined at its carboxyl-terminal end to various rSkM1 or
rSkM2 cytoplasmic segments were prepared. Fusion proteins containing
the rSkM1 or rSkM2 carboxyl termini, the rSkM1 interdomain 2-3
region, or MBP alone were immobilized on amylose resin and incubated
with a synthetic peptide corresponding to residues 1-30 of the
rSkM1 amino acid sequence (1-30 peptide), the region containing
the A/B2 and L/D3 epitopes. Bound peptide was identified by subsequent
incubation of the peptide-fusion protein-resin complex with either A/B2
or L/D3 followed by We
found no specific binding of the 1-30 peptide to fusion proteins
containing the rSkM2 carboxyl terminus, the rSkM1 interdomain
2-3, or the MBP alone. However, the peptide bound specifically
and with high affinity to the rSkM1 sodium channel carboxyl-terminal
fusion protein (Fig. 5).
Figure 5:
Binding of I 1-30 peptide to fusion
proteins containing different sodium channel intracellular domains.
Fusion proteins composed of maltose-binding protein linked to the rSkM1
carboxyl terminus (residues 1593-1840), the rSkM1 interdomain
2-3 region (residues 794-1017), the rSkM2/rH1 carboxyl terminus
(residues 1791-2018), and the maltose binding protein alone were
immobilized on amylose resin and incubated with a synthetic peptide
comprising residues 1-30 of the rSkM1 sodium channel amino
terminus (I 1-30). This peptide bound specifically and with high
affinity only to the rSkM1 carboxyl-terminal fusion protein, providing
experimental support for the hypothesis that these two channel segments
interact in vivo.
Based on previous estimates(18) , the absence of
competition between two antibodies for binding suggests that their
epitopes are located more than 3.5 nm apart or are constrained to face
in opposite directions. True competitive binding occurs when the
epitopes are in close physical proximity. For intermediate separations
( For most
monoclonal-polyclonal pairs, maximum competition decreased specific
binding to It is possible that some
of the purified channel protein may be sufficiently denatured to allow
separation of epitopes, even though we have shown in the past that most
remain functional(16, 17) . However, since some
monoclonal-polyclonal pairs produce greater binding inhibition
(>80%), it is more likely that the lower levels of inhibition seen
with other pairs simply reflect incomplete block as expected for
intermediately separated epitopes where multiple polyclonal antibody
molecules with varying affinities may need to bind in order to
completely occlude the second epitope. Finally, we have no data that
addresses the possible role of alternate channel conformations or
variable post-translational modification in this process.
Figure 6:
Cartoon depicting our model of sodium
channel cytoplasmic domain structure. The figure on the left reflects the view obtained from inside the cell, looking up at the
portions of the sodium channel protein which extend into the cytoplasm.
The figure on the right represents a side view of the sodium
channel protein. Specific points to observe include: (a) the
interaction of the carboxyl terminus (lightest shade of gray)
with the arch-shaped amino terminus (darkest shade of gray),
presenting a face extending away from the bulk of the intracellular
mass of the channel; (b) the centrally located interdomain
2-3 region with a fixed orientation to the remainder of the
channel's intracellular segments; (c) the relative
organization of each antibody epitope in three-dimensional space. See
text for details.
Our data
support a model in which the first 46 amino acids of the amino terminus
forms an arc, with the A/B2 epitope (residues 1-6) facing away
from, the L/D3 epitope (residues 19-24) facing partly toward, and
the I-31 epitope (residues 31-46) facing directly toward the
centrally located interdomain 2-3 region (Fig. 6).
Evidence supporting this hypothesis includes the absence of interaction
between A/B2 and all polyclonal antibodies except I-1771, the partial
interaction of L/D3 with B-30 (interdomain 2-3) and B-23
(beginning of carboxyl terminus), and the interaction between I-31 and
monoclonal antibodies F/C11 and B/D6 (interdomain 2-3 region).
We have
shown that A/B2 and L/D3 demonstrated competition with each other for
binding to immobilized sodium channel protein(11) . However,
unlabeled F/C11 competed with neither A/B2 nor L/D3 while unlabeled
B/D6 demonstrated partial competition with A/B2 and L/D3. No
competition was observed between labeled F/C11 and unlabeled B/D6.
These data are consistent with our topologic model of the sodium
channel amino terminus and interdomain 2-3 regions. The
absence of competition between monoclonals to two adjacent interdomain
2-3 epitopes (F/C11 and B/D6) indicates that these two epitopes
are oriented in different directions and, although separated by
approximately 100 residues, may have a restricted range of motion. The
observation that this region is the most sensitive to proteolysis of
the interdomain regions supports a model in which the amino-terminal
half of the interdomain 2-3 region extends into the cytoplasm,
away from the membrane embedded domains (Fig. 6). The partial
inhibition by A/B2 and L/D3 on B/D6 binding, the absence of inhibition
by these two monoclonal antibodies on either B-30 or F/C11 binding, and
the competition between I-31 and both F/C11 and B/D6 all suggest that
the B/D6 epitope (located at the carboxyl-terminal end of the
interdomain 2-3 region) is oriented toward and/or is closer to
the amino terminus than are the F/C11 or B-30 epitopes (located at or
just beyond the mid-portion of the interdomain 2-3 region) (Fig. 6). Testing these structural hypotheses will require
further dissection of cytoplasmic domain topology using competition
studies with antibody Fab fragments, physical probes such as
fluorescence energy transfer, and direct imaging techniques such as
electron diffraction.
Volume 270,
Number 38,
Issue of September 22, pp. 22271-22276, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR THE INTERACTION OF THE rSkM1 AMINO AND CARBOXYL
TERMINI (*)
Materials
Materials for the preparation of
oligopeptides and antibodies, the isolation of crude membranes, and for
the purification of membrane proteins were obtained from sources
previously identified(8) . DEAE-Sephadex (A25-120), wheat germ
agglutinin-agarose, protease inhibitors, and one set of prestained
molecular mass standards (26.2-180 kDa) were obtained from Sigma.
Another set of prestained molecular mass standards (15-110 kDa)
was obtained from Bio-Rad. I-protein A and
I-labeled goat anti-mouse IgG were from ICN
Radiochemicals (Irvine, CA). The pMal fusion protein and purification
system was obtained from New England Biolabs.
Preparation of Antisera
Monoclonal antibodies were
previously generated by immunizing mice with purified
detergent-solubilized rat skeletal muscle sodium channel
protein(13) . Oligopeptides were synthesized and polyclonal
antibodies generated against these synthetic oligopeptides using
methods detailed in earlier
publications(8, 9, 10) . All oligopeptides
correspond to regions of the rat skeletal muscle sodium channel protein
sequence ( Fig. 1and Table 1). The carboxyl-terminal
cysteine residue which terminates each peptide is not part of the
naturally occurring sequence and was added to assist in coupling
peptide to carrier protein prior to rabbit immunization.
Sodium Channel Protein Purification
Preparation of
muscle surface membranes containing unproteolyzed sodium channels was
identical to that described previously(8) . Sodium channel
protein was solubilized using Nonidet P-40 and then purified using
sequential ion exchange and lectin affinity chromatography(8) .
Purified solubilized sodium channel protein was kept at 4 °C and
used within 2 weeks.Antibody Binding Assay
Purified rSkM1 protein (5
pmol) was adsorbed to 25 µl of wheat germ lectin-Sepharose 4B by
gentle agitation for 1 h at room temperature. The resin was pelleted
and washed with phosphate-buffered saline, pH 7.4 (PBS). (
)Monoclonal or polyclonal antisera was added, incubated
with resin, and samples were removed at 30-min intervals over 6 h.
After pelleting and washing, the resin was incubated with 5 µCi of I-labeled goat anti-mouse IgG (monoclonal antibodies) or
5 µCi of
I-protein A (polyclonal antibodies) for 2 h
at room temperature. The resin was then washed with PBS and immobilized
radioactivity quantitated in a gamma counter.
Antibody Competition Assay
Purified rSkM1 protein
(5 pmol) was added to 25 µl of wheat germ lectin-Sepharose 4B and
incubated for 1 h at room temperature. The resin was gently pelleted
and washed with PBS. Serial dilutions of polyclonal antibody were added
and incubated with resin for 2 h. The resin was washed again and then
incubated with a fixed concentration of secondary antibody (monoclonal
supernatant) for 4 h at room temperature. Following pelleting and
washing, the resin was incubated with 5 µCi of I-labeled goat anti-mouse IgG for 2 h at room temperature
and washed with PBS; immobilized radioactivity was quantitated in a
gamma counter.
Fusion Protein Constructs
Fusion proteins
expressed from subcloned fragments of the rSkM1 and rSkM2 (also known
as rH1) interdomain 2-3 or carboxyl-terminal regions were
expressed and purified using the pMal protein fusion and purification
system (New England Biolabs). Briefly, the cloned gene was inserted
downstream from the malE gene encoding the maltose-binding
protein (MBP). This construct directs the expression of a fusion
protein containing MBP at its amino-terminal end and a fragment of the
cloned sodium channel interdomain or carboxyl-terminal region at the
carboxyl-terminal end. Details of construct synthesis and the methods
used to generate different length fragments of the rSkM1 interdomain
2-3 and rSkM1 and rSkM2 carboxyl-terminal regions are given in
another publication(14) . Western blots of these fusion
proteins incubated with monoclonal antibodies F/C11 and B/D6 were used
to localize the epitopes for these antibodies (see Sun et al.(14) for details).Peptide Binding to Interdomain 2-3 or
Carboxyl-terminal Fusion Proteins
Fusion protein constructs (0.5
nmol) bound to amylose resin were incubated with 50 nmol of synthetic
peptide for 4 h at room temperature or overnight at 4 °C. Resin was
washed five times with PBS, 0.25% Tween 20, incubated with either A/B2
or L/D3 monoclonal supernatant (dilution 1:10) for 4 h, washed again as
above, and then incubated with 0.8 µCi of I-labeled
goat anti-mouse IgG for 4 h at room temperature or overnight at 4
°C. The resin was washed five times with PBS, 0.25% Tween 20 over 1
h before radioactivity bound to resin was quantitated by gamma
counting.
Monoclonal Epitope Localization
Having localized
the epitopes for representative members of one group of monoclonal
antibodies (A/B2 and L/D3) to the amino terminus of rSkM1 in a previous
study(12) , our initial goal was to identify the epitopes for
two representative members (F/C11 and B/D6) of a second group of
monoclonal antibodies. Regional localization was obtained by comparing
the pattern of proteolyzed rSkM1 sodium channel fragments that bound
these antibodies to the patterns already described for sodium channel
fragments visualized by antibodies directed against known epitopes
distributed throughout the channel primary structure(8) . The
pattern of fragments as well as the limit peptide identified by
antibodies F/C11 and B/D6 indicates that the epitopes for these two
monoclonal antibodies lie within a region encompassing interdomain
2-3 and domain 3 (Fig. 2).
-chymotrypsin (0.5
µg/ml) at room temperature(8) . All antibodies identify the
276-kDa subunit in the nonproteolyzed samples. The pattern of the
limit digest indicates that the epitopes for the two unknown monoclonal
antibodies (F/C11 and B/D6) are located on the same
limit fragments as the epitope for B-30 (i.e. interdomain
2-3 and domain 3).
Antibody Competition Studies
To investigate the
spatial relationships of sodium channel intracellular segments, four
representative monoclonal antibodies were assayed for binding
competition with a panel of polyclonal antisera for which defined
epitopes are already known (Table 1). Measurements were made with
solubilized purified rSkM1 sodium channel protein in a mixed micellar
form that retains native channel structure, as indicated by toxin
binding activity(15) , sensitivity to proteolytic
enzymes(8) , and capacity for functional
reconstitution(16, 17) . Channel protein was
immobilized to wheat germ-Sepharose beads. We first measured the
kinetics of antibody binding to immobilized sodium channel protein to
ensure that all measurements were performed under equilibrium
conditions. Binding of individual monoclonal antibodies to the channel
reached 90% of final values within 15 min of incubation at 22 °C.
When no competition was seen between a test monoclonal antibody and a
polyclonal antiserum, polyclonal antibody binding prior to the addition
of monoclonal antibody had no effect on the kinetics of monoclonal
antibody binding. As expected, when competition between antibodies was
observed, prior incubation with a polyclonal antibody frequently slowed
the rate at which a subsequently applied monoclonal antibody bound,
although in all cases binding appeared to reach equilibrium within four
hours at 22 °C.
I-labeled goat anti-mouse IgG.
3.5 nm), one antibody may sterically hinder the approach of a
second to its epitope while not preventing its ultimate binding. Since
conformational changes induced by the binding of one antibody that
reduce the affinity of another antibody at a remote epitope appear to
occur infrequently(18) , we are able to place constraints on
the organization of the epitopes under study within the roughly 9-nm
diameter envelope of the sodium channel protein (see Barchi (19) for a review of channel physical properties).
35% of control values even with the highest
concentrations of competing antisera. While this could reflect a
measuring artifact resulting from a reduction in k
for the second antibody, this is not the case here since binding
rates were determined directly and measurements were made under
equilibrium conditions. Several other explanations must be considered.
First, a portion of the solubilized sodium channel protein used in the
binding assay may be denatured during preparation or storage, resulting
in the spatial separation of epitopes which otherwise are close
together. Alternatively, sodium channel protein may exist in different
conformations, only some of which position the epitopes close to one
another. Finally, variable post-translational modification of the
sodium channel protein (e.g. phosphorylation) may prevent
quantitative monoclonal antibody binding.
Predictions Concerning the Amino Terminus
Several pieces
of data suggest a rigid structure for the amino terminus (Fig. 6). These include: 1) the absence of competition between
I-31 and either A/B2 or L/D3 despite the fact that all three epitopes
are located within the first 46 residues of the channel sequence; 2)
the lack of competition between A/B2 and all site-directed polyclonal
antibodies except I-1771 (see below); and 3) the relative resistance of
the amino terminus to exogenous proteolysis(8) .
Predictions Concerning the Interdomain 2-3
Region
All of our polyclonal antibodies interfere with
monoclonal antibody binding to the interdomain 2-3 region,
suggesting that this region is centrally located on the cytoplasmic
surface of the channel within a 3-3.5 nm distance from each of
the polyclonal epitopes (Fig. 6). In our proteolysis study, the
interdomain 2-3 region, while not the largest, was the most
accessible of the interdomain regions to exogenous proteolysis using
soluble proteases(8) , consistent with a model in which the
interdomain 2-3 region extends away from the membrane embedded
portion of the channel into the cytoplasmic aqueous phase.Binding of Amino to Carboxyl Terminus
Our data
suggest that the channel amino and carboxyl termini are closely
interrelated in the tertiary structure of the channel. In order to
accommodate this interaction, we suggest a model in which domains 1 and
4 are adjacent in the tertiary structure. This is consistent with
previous binding studies which demonstrated competition between
scorpion toxin and site-specific antibodies directed against the S5-S6
regions of domains 1 and 4 in the rat brain sodium
channel(20) . Although the functional significance of this
interaction is unknown, binding between the amino and carboxyl termini
could play a role in channel assembly or in stabilization of channel
tertiary structure. Another possibility is suggested by our previous
finding that A/B2 and L/D3 differentially label channels in the surface
and T-tubular membranes of fast and slow skeletal muscle
fibers(21, 22) . It is possible that the rigid surface
formed by the interaction of amino- and carboxyl-channel segments
creates a binding site for cytoskeletal proteins that contribute to the
subcellular localization of channels in different membrane
environments(12) .
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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