(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--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).
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 I-labeled goat anti-mouse IgG.
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
(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).
For most
monoclonal-polyclonal pairs, maximum competition decreased specific
binding to 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.
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.