From the Department of Cell Biology and Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received for publication, March 31, 2003 , and in revised form, April 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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Polarized localization of VGSCs in the axonal membrane requires interactions with members of the ankyrin family of peripheral membrane proteins (reviewed in Ref. 7). Ankyrins function as membrane-cytoskeleton adaptors that immobilize integral membrane proteins to the spectrin-based membrane skeleton. Ankyrins co-purify with and directly bind to purified VGSCs (8), and specific isoforms of the ankyrinG gene are concentrated with VGSCs at the IS (9), the nodes of Ranvier (9), and the neuromuscular junction (10). The functional importance of the interaction between ankyrinG and VGSCs is demonstrated in knockouts of ankyrinG in the mouse cerebellum. Purkinje cells from knockout animals are deficient in localized VGSC concentrations at the IS (11) and are unable to initiate action potentials (12).
In addition to VGSCs, ankyrins also interact with a variety of other integral membrane proteins present at the node including the CAMs neurofascin and NrCAM (13, 14). Interactions of ankyrins with VGSCs (15) and other integral membrane proteins are mediated through the N-terminal 90-kDa repeat or membrane-binding (MB) domain. The interaction between the ankyrin MB domain and neurofascin/NrCAM is dependent upon a conserved FIGQY motif in the cytoplasmic domain of these molecules (16).
Unlike neurofascin and NrCAM, the nature of the interaction between VGSCs
and ankyrins remains unclear. Initial studies using uncharacterized rat brain
preparations of VGSCs and purified erythrocyte ankyrin
(15) did not distinguish which
VGSC subunits or their isoforms were involved in ankyrin binding. Subsequent
studies using a variety of techniques have suggested sites for ankyrin
interaction on both the VGSC
(17) and
subunits
(18).
In this study, we identified a domain of the VGSC- subunit that
interacts with ankyrinG and demonstrated its role in the
localization of VGSCs to the IS in vivo. Initially we observed that
rNav1.2 (rat Nav1.2) co-localizes with
ankyrinG at the IS of hippocampal neurons and identified an
ankyrinG-binding site in the cytoplasmic linker between domains II
and III (loop 2), using a pull-down assay. We did not detect binding sites for
ankyrinG elsewhere within the
subunit nor did we detect
binding with the
subunits using this assay. We further identified a
9-aa motif within the loop 2 sequence that is conserved among all vertebrate
subunits and demonstrated its requirement in ankyrinG
binding. Finally, we demonstrated that deletion of this 9-aa motif abolishes
the ability of neurofascin/loop 2 chimeric molecules to be localized to the IS
of cultured hippocampal neurons. These results demonstrate that ankyrin
binding is conserved among vertebrate sodium channels and is crucial to the
localization of VGSCs to excitable membrane domains.
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EXPERIMENTAL PROCEDURES |
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Immunofluorescence Analysis of Primary Hippocampal Neurons For immunostaining of primary hippocampal neurons, coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and processed as described (19). Primary antibodies used were rabbit anti-Nav1.2 (1:100; Upstate Biotechnology, Inc., Lake Placid, NY), or mouse anti-Myc (1:1000; Covance, Berkeley, CA), and chicken anti-ankyrinG 480/270 kDa (1:2000; (14)). For neurons transfected with neurofascin/loop 2 chimeras, cells were incubated with a mouse anti-HA antibody (1:2000; Covance) before permeabilization and staining with the chicken anti-ankyrinG 270/480-kDa antibody. Bound antibodies were detected by incubation for 1 h at 37 °C with Alexa 594-nm conjugated goat anti-rabbit or goat anti-mouse and Alexa 488 nm goat anti-chicken (Molecular Probes, Inc., Eugene, OR). Fluorescent images were captured on a Leica DM IRE2 inverted microscope (Leica Microsystems, Inc., Bannockburn, IL) using an ORCA ER extended-range cooled CCD camera (Hamamatsu Photonics, Inc.) and Openlab software (Improvision, Inc., Lexington, MA).
Construction of Plasmids for Protein ExpressionRat cDNAs
for the 1 and
2 subunits of the voltage-gated sodium channels (a
gift from Dr. L. L. Isom, University of Michigan) were subcloned into pEGFP-N1
(BD Biosciences) (not in frame with the downstream GFP), and a sequence coding
for the HA epitope (YPYDVPDYA) was inserted by PCR at a position corresponding
to aa 26 for
1 and 34 for
2 (see
Fig. 1b). Constructs
were verified by sequencing.
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cDNA constructs encoding combinations of different domains of the
rNav1.2a molecule (full-length cDNA for rNav1.2a was a
gift from Dr. A. L. Goldin, University of California, Irvine, CA) fused to the
C terminus of GFP (see Figs. 1
and 2) were obtained using PCR
and standard molecular biology techniques. Similarly, PCR was used to generate
a series of chimeric channels (see Fig.
3) encompassing areas of loop 1 from rNav1.2a (aa
599694, construct II-1-III) and loop 2 from rNav1.1 (aa
10331131, construct II-III (1.1)), rNav1.6 (aa
10141108, construct II-III (1.6)), rNav1.4 (aa
837933, construct II-III (1.4)), and rNav1.5 (aa
9831102, construct II-III (1.5)). These substitutions were based on a
sequence alignment of the whole loop 2 region of these isoforms with
rNav1.2a (see Fig.
2c). Rat cDNAs for Nav1.1, Nav1.6,
and Nav1.4 were gifts from Dr. A. L. Goldin, Dr. J. H. Caldwell
(University of Colorado Health Sciences Center, Denver, CO) and Dr. J. S.
Trimmer (State University of New York, Stony Brook, NY), respectively. A
partial cDNA for rNav1.5 was obtained by reverse transcriptase-PCR
using the Prostar kit (Stratagene, La Jolla, CA) on rat heart mRNA isolated
with the Oligotex kit (Qiagen, Inc., Chatsworth, CA) following the
manufacturers' specifications. Deletion of consensus sequence
VPIAXX-ESD in constructs II-III and II-III (1.5) was obtained by
"loop-out" mutagenesis using the QuikChange kit (Stratagene)
following the manufacturer's instructions (constructs II-III and
II-III
(1.5), respectively; see Fig.
3).
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Neurofascin/Nav Chimeric
MoleculesFull-length cDNAs encoding HA-tagged neurofascin 186-kDa
isoforms were a gift from Dr. Vann Bennett (Duke University, Durham, NC), and
their preparation has already been described
(20). The 500-bp
ApaINotI fragment representing the cytoplasmic domain
of neurofascin was subcloned into pBlue-script (Stratagene), and the Y81A
mutation was generated using the QuikChange mutagenesis kit (Stratagene)
before the altered cytoplasmic domain fragments were re-introduced into
full-length neurofascin molecules. PCR products representing the loop 2
sequence from Nav1.2a and -1.2 (Nav1.2a missing
the VPIAXXESD motif) were modified to include a 5' in-frame
ApaI site and a 3' stop codon upstream of a NotI site.
These products were then substituted for the neurofascin-cytoplasmic domain in
full-length HA-tagged neurofascin (NF) 186-kDa molecules to generate
neurofascin/Nav chimeric molecules (see Figs.
3 and
4).
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Sindbis PseudovirionsPCR was used to generate a 3'
fusion of the 24-ankyrin repeats from rat ankyrinG (MB, aa
1853) with sequence-encoding glutathione S-transferase (GST).
The cDNA for MB-GST, or GST alone, was cloned into the pSinRep5 vector
(Invitrogen) and used as a template to synthesize capped RNA transcripts
in vitro with the Invitroscript CAP kit (Invitrogen). The transcripts
were electroporated into BHK cells along with similarly prepared transcripts
encoding the structural genes for the DH(26S) helper phage. BHK cells were
then cultured in -MEM with 5% fetal bovine serum for 48 h, and the
media containing Sindbis pseudovirions for MB-GST or GST were collected,
aliquoted, and kept at80 °C.
In Vitro AnkyrinG Binding
AssayMedia enriched with Sindbis pseudovirions for MB-GST or GST
were diluted in serum-free -MEM, and optimal viral dilutions were
determined to obtain high and reproducible levels of protein expression. 450
µl of these dilutions were applied to
106 BHK cells in 60-mm
dishes and incubated for 1 h at 4 °C with intermittent rocking. Then 4
ml/dish
-MEM with 1% FBS (v/v) was added, and the cells were incubated
for a further 24 h at 37 °C. Cells were then washed twice in
phosphate-buffered saline and incubated for 20 min on ice with 600 µl/dish
lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1% Triton
X-100, 0.05% Tween 20, 1 mM Na2EDTA, pH 8.0) in the
presence of protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml each antipain, leupeptin, and pepstatin). Lysates were
centrifuged, and the supernatant containing MB-GST or GST was incubated with
one-fifth volume of glutathione-Sepharose 4B in a 50% slurry (Amersham
Biosciences) for 30 min at 4 °C on a rocker. The beads were then washed
three times in lysis buffer and kept on ice before use.
For pull-down experiments with MB-GST or GST alone as a control, BHK cells were transfected with GFP-Nav or neurofascin/Nav chimeric constructs as described above and incubated for 24 h at 37 °C. Cells were then lysed as above in 600 µl/60-mm dish of lysis buffer with protease inhibitors. 300 µl of cell lysate was then added to either 20 µl of MB-GST beads or 20 µl of GST beads and rocked overnight at 4 °C. The concentration of MB-GST or GST used in each pull-down was typically 2 µM. After incubation, the beads were washed three times with ice-cold lysis buffer, and bound proteins were eluted twice with 1 bed volume of 10 mM reduced glutathione in 50 mM Tris-Cl, pH 8.0. Cell lysates and proteins associated with MB-GST and GST were probed by immunoblotting using antibodies to GFP (Covance) or the HA tag (Covance). Similarly, cell lysates were analyzed to ensure expression of the tagged protein, and batches of beads used in the assays were analyzed using antibodies to GST to ensure the presence of the MB-GST and GST proteins (not shown).
Yeast Two-hybrid SystemThe MB domain from ankyrinG was subcloned in frame with the Gal4 activation domain into LEU2-marked prey vector pGAD424 (a gift from Dr. Peter Pryciak, University of Massachusetts Medical School, Worcester, MA). The loop 2 EcoRI cassettes described above were subcloned in frame with the LexA DNA-binding domain into TRP1-marked bait vector pBTM116. The cytoplasmic domain of neurofascin in its wild type form or carrying the Y81A mutation was also subcloned into pBTM116 to be used respectively as the positive or negative control for interaction with MB. Yeast strain L40, harboring the HIS3 reporter gene under the control of LexA-binding sites, was co-transformed with 50 ng each of the bait and prey vector using the lithium/acetate method and then streaked out onto agar plates lacking tryptophan and leucine. For each transformation, six independent clones were selected and replica-plated onto media lacking tryptophan, leucine, and histidine to assess expression of the HIS3 reporter gene.
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RESULTS |
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We developed a pull-down assay to determine ankyrinG-binding
sites within defined subunits and domains of rNav1.2a using a GST
fusion of the repeat domain (MB) of ankyrinG (MB-GST). This
construct was expressed at high levels in BHK cells using the Sindbis viral
expression system and immobilized on glutathione-Sepharose beads. Similar
beads coupled to GST alone were used as controls for each assay. To test for
interactions with subunits, beads were added to cell lysates from BHK
cells expressing HA-tagged
1 and
2 subunits
(Fig. 1b). As shown in
Fig. 1b, these
subunits did not exhibit interactions with MB-GST.
To test for interactions with the rNav1.2a subunit, we
incubated MB-GST and GST beads with BHK cell lysates expressing full-length
rNav1.2a. However, we were unable to obtain efficient
solubilization of the full-length rNav1.2a protein under the
non-denaturing conditions used in our binding assay (data not shown). We
therefore generated a series of GFP-tagged constructs that spanned
rNav1.2a (Fig.
1c) in which we tried to maintain the structure of the
cytoplasmic loops while preserving the integrity of the transmembrane domains
(Fig. 1c). Constructs
spanning the rNav1.2a
subunits III-IV, II-III, and the III
domain all precipitated with MB-GST beads and not with GST beads alone
(Fig. 1c). This
interaction did not appear to involve the C terminus of rNav1.2a as
deletion of this region of the molecule from construct III-IV (construct
III-IV
C) did not interfere with its ability to interact with MB-GST. In
contrast, domain I-II of rNav1.2a and a GFP fusion of the
cytoplasmic C terminus alone (not shown) did not exhibit interactions with
MB-GST.
These results suggested the presence of a site of ankyrinG interaction between aa 1044 and 1474 of rNav1.2a. Further studies in which individual cytoplasmic loops of the III-IV construct were replaced sequentially with the nonbinding N terminus of rNav1.2a confirmed that the binding site was restricted to loop II-III (not shown). These results are in contrast to the findings of Bouzidi et al. (18) who recently reported interactions between ankyrinG and fusion proteins representing the isolated intracellular loops between domains I-II and III-IV but not between the II-III loop and ankyrinG.
AnkyrinG Associates with a Conserved 9-aa
Motif in Loop 2 of VGSC- SubunitsTo further define
the ankyrinG-binding site within loop 2, we used deletion analyses.
An internal deletion of aa 10241121 in loop 2 or its substitution with
a portion of loop 1 (aa 599694)
(Fig. 2a, constructs
II-
-III and II-1-III, respectively)
abolishes the interaction with MB-GST restricting the binding site to aa
10441121 of rNav1.2a.
Fig. 2c shows an alignment of the sequence of loop 2 between rat VGSCs. A high degree of homology is observed between aa 10241121 of rNav1.2a and the analogous region of other neuronal isoforms (87, 76, 71, and 65% identity with rNav1.1, -1.3, -1.6, and -1.7, respectively) with the exception of rNav1.8 and rNav1.9 (only 15 and 26% identity, respectively). This region is less well conserved between rNav1.2a and the skeletal muscle and cardiac isoforms rNav1.4 (41% identity) and rNav1.5 (16% identity), respectively.
We decided to exploit the range of conservation within various loop 2 sequences to further map the site of ankyrinG interaction. We substituted aa 10241121 in construct II-III with the analogous region (Fig. 2c) of rNav1.1, rNav1.6, rNav1.4, and rNav1.5 to generate four chimeras (II-III (1.1), II-III (1.6), II-III (1.4), and II-III (1.5)). These constructs were expressed in BHK cells and used in pull-down assays with MB-GST as before. Chimeras II-III (1.1), II-III (1.6), and II-III (1.4) were specifically precipitated by MB (Fig. 2b). Despite its low level of homology, chimera II-III (1.5) also retained the ability to bind to MB (Fig. 2b). Careful analysis of the sequence of these five isoforms revealed only one stretch of nine amino acids conserved in this region, VPIAXXESD (indicated in bold in Fig. 2c), corresponding to aa 11051113 in rNav1.2a and located within the binding region defined previously.
We selectively deleted this sequence of 9 aa in constructs II-III and
II-III (1.5) using loop-out mutagenesis to obtain the deletion mutants
II-III and II-III
(1.5), respectively. As shown in
Fig. 2d, deletion of
this 9-aa motif resulted in a loss of binding to MB-GST. The sequence
VPIAXXESD is thus necessary for association of both II-III and II-III
(1.5) with MB and defines a binding sequence for ankyrinG within
the
subunit rNav1.2a.
Fig. 2e shows that
this motif is conserved among rat VGSCs regardless of the overall degree of
homology in loop 2. We have found that its presence is associated with binding
to MB-GST in at least six isoforms (indicated with a "+" in
Fig. 2e) including the
lesser conserved motif observed in rNav1.9 (APLAEVEDD, not shown).
The sequence (V/A)P(I/L)AXXE(S/D)D thus defines a conserved motif in
the
subunits of VGSCs involved in the binding of
ankyrinG.
To test whether loop 2 and the conserved 9-aa motif were sufficient and
necessary for ankyrinG association, we constructed chimeric
molecules consisting of HA-tagged neurofascin extracellular domain fused to
the loop 2 sequence (Fig.
3a). Loop 2 sequences from rNav1.2a and a
variant of rNav1.2a lacking the 9-aa motif
(rNav1.2) were utilized
(Fig. 3a). These
chimeric constructs along with wild type neurofascin 186 kDa were used in
pull-down assays with MB-GST. As a negative control, we mutated the tyrosine
residue within the FIGQY motif of wild type neurofascin to an alanine (NF
Y81A). Phosphorylation of this tyrosine residue has been previously shown to
abolish ankyrin binding
(20).
Fig. 3a shows that
chimeric molecules containing loop 2 sequences from rNav1.2a (NF
1.2) exhibited interactions with MB-GST suggesting that loop 2 alone was
sufficient to promote association with ankyrinG. However, deletion
of the conserved 9-aa motif (NF 1.2) led to a loss of MB-GST
interaction. As expected, wild type neurofascin associated strongly with
MB-GST, whereas mutation of the tyrosine residue to alanine (NF Y81A)
abolished this interaction despite comparable expression levels
(Fig. 3a). The
observation that a single aa change can abolish ankyrin binding illustrated
the sensitivity and specificity of our pull-down assay using MB-GST. Our data
demonstrate that loop 2 and the conserved 9-aa motif are necessary for
ankyrinG binding.
To determine whether the loop 2/ankyrinG interaction involved
proteins specific to BHK cell lysates, we carried out a yeast two-hybrid
reaction. As shown in Fig.
3b, the loop 2 sequence from rNav1.5 fused to
the LexA DNA-binding domain (pBTM Nav1.5) was able to activate
expression of the L40 HIS3 reporter gene when co-transfected with the
MB sequence fused to the Gal4 activation domain (pGAD MB). His3 was not
expressed when pBTM Nav1.5 was co-transfected with the Gal4
activation domain alone nor when a mutated version of rNav1.5
lacking the 9-aa motif (pBTM Nav1.5) was used. The wild type
neurofascin cytoplasmic domain and the Y81A variant of this domain were used
as positive and negative controls, respectively
(Fig. 3b). Similar
studies involving loop 2 sequences from Nav1.2 and -1.6 could not
be carried out as these constructs were observed to transactivate reporter
genes in the absence of pGAD MB.
The Conserved 9-aa Motif Affects Localization to the IS of Cultured
Hippocampal NeuronsTo determine whether loop 2 and the 9-aa motif
affected localization of membrane proteins at the IS, we utilized the
neurofascin/loop 2 chimeric constructs. These molecules were transfected into
3 days in vitro hippocampal cells, and these cells were fixed and
stained with HA antibodies at various time points after transfection to
determine when and where these proteins could be first detected on the cell
surface. Cells were then permeabilized and stained with antibodies to
ankyrinG 480/270 kDa to delineate the IS. As shown in
Fig. 4, 75% of neurons 6 h
after transfection with neurofascin/loop 2 chimeric molecules (NF 1.2)
exhibited concentrated HA staining at their IS
(Fig. 4A) as
delineated by ankyrinG staining
(Fig. 4A,
inset). Staining was also observed on the cell body of transfected
neurons. 24 h after transfection,
90% of the neurons now exhibited HA
concentrations at the IS, and neuronal cell body staining was diminished
compared with the signal at the IS (Fig.
4E). In contrast to wild type loop 2 molecules, chimeric
molecules lacking the 9-aa motif (NF 1.2
) were observed over the
surface of the entire neuron and did not exhibit concentration at the IS even
at later time points (Fig. 4, B
and F). Indeed, at earlier time points NF 1.2
showed increased concentrations at the distal end of the axon
(Fig. 4B).
We also introduced wild type and Y81A neurofascin constructs into hippocampal neurons. Unlike NF 1.2, wild type neurofascin did not immediately exhibit concentrations at the IS except in a small population of neurons (<10%). Instead, HA staining was distributed throughout the cell (Fig. 4G) in a similar fashion to that observed for the Y81A mutant (Fig. 4D). However, NF was observed to accumulate over time at the IS, such that between 45 and 65% of transfected neurons showed IS accumulations of neurofascin at 24 h after transfection (Fig. 4G). Similar concentrations were not observed for the Y81A mutation (Fig. 4H) supporting the idea that concentration of chimeric constructs at the IS is independent of the neurofascin extracellular domain. These results indicate that the 9-aa motif is necessary for the specific localization of neurofascin/loop 2 chimeric constructs at the IS.
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DISCUSSION |
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Recent studies have yielded conflicting data on the nature of the
interaction between ankyrin and VGSCs. Regulated interactions between ankyrin
and VGSC- subunits have been detected using a cell-based recruitment
assay (17,
21). In contrast to these
results, Bouzidi et al.
(18) did not detect
interactions between ankyrinG and the cytoplasmic domains of the
subunits using bacterially expressed proteins in an in vitro
binding assay. However, they did detect interactions with recombinant fusion
proteins representing the I-II (loop 1) and the III-IV (loop 3) cytoplasmic
linkers. They detected no interactions with loop 2. In our study, we also did
not detect interactions with the VGSC-
subunits using
ankyrinG, but detected an interaction with the loop 2 cytoplasmic
linker of the
subunit. We further confirmed this interaction using
yeast two-hybrid and demonstrated that loop 2 was sufficient for the
co-localization of chimeric molecules at the IS with ankyrinG.
Differences between our findings and those of Bouzidi et al.
(18) might reflect their use
of bacterially expressed polypeptides. In support of this idea, we have been
unable to detect interactions between bacterially expressed
ankyrinG and loop 2 in an in vitro assay.
To further examine the effects of loop 2 on VGSC- subunit
localization we have used chimeric neurofascin/Nav1.2 molecules and
observed that these molecules localize at the IS in the presence of the 9-aa
motif. The observation that chimeric molecules are also initially detected on
the neuronal cell body but are down-regulated there over time
(Fig. 4) suggests that the
retention of chimeric molecules at the IS is important for their
concentration. However, ankyrin binding could also play a role in the
trafficking of these chimeric molecules to the initial segment as has been
suggested for the intracellular sorting of
Ca2+-homeostasis proteins in cardiomyocytes
(22).
The finding of a conserved motif in VGSCs involved in ankyrinG interaction is supported by increasing evidence for spatial and temporal diversity in VGSC isoforms enriched at ankyrinG-defined excitable membrane domains. Both Nav1.1 and Nav1.2 have been observed at the nodes of Ranvier in young mice and are gradually replaced with Nav1.6 during development (23, 24). Nav1.6 is the VGSC isoform found at most adult nodes in the peripheral nervous system and the central nervous system (25); however, Nav1.2, -1.8, and -1.9 have also been detected at adult nodes (24, 26, 27). A similar diversity is observed at the IS where Nav1.2, -1.6, and -1.8 have been localized (11, 26). In other tissues, ankyrinG is also localized to the neuromuscular junction (10) where it is a candidate to interact with skeletal isoforms of the sodium channel such as Nav1.4. The observation that ankyrinG can also interact with cardiac Nav1.5 suggests that isoforms of ankyrinG might also be linked with heart disease as has been shown for ankyrinB in Long QT syndrome (28).
What are the mechanisms responsible for the segregation of specific VGSC isoforms with ankyrinG in excitable membrane domains? The distinct spatial and temporal pattern of expression of VGSC isoforms in nervous tissue (29) is clearly a factor in determining the molecular identity of the VGSC present at the nodes of Ranvier and the IS. Indeed, the gradual isoform switch from Nav1.2 to Nav1.6 at developing nodes is paralleled by a down-regulation of Nav1.2 and an up-regulation of Nav1.6 proteins (24). The selective segregation of VGSCs with ankyrinG could also result from isoform-dependent differences in the affinities of interaction that are not detectable by the methods used in this study. In addition, post-translational modifications similar to those reported for neurofascin (20) could regulate the ankyrin/VGSC interaction and play a role in determining VGSC specificity.
Additional sequences within the subunit could also influence
localization of specific VGSC isoforms within neurons. For example, the C
terminus of rNav1.2 targets CD4 chimeric molecules to the axon of
hippocampal neurons, whereas distribution of a similar construct derived from
the C terminus of rNav1.6 is restricted to the somatodendritic
region (30). The role of
accessory subunits in VGSC localization should also be considered.
subunits appear to influence the surface expression of VGSC isoforms
(31) and are suspected to
exhibit selective associations with different
subunits
(24). Similarly, the
association of annexin II light chain with the N terminus of Nav1.8
increases its translocation to the plasma membrane
(32). Thus, accessory subunits
may participate in the functional localization of VGSC isoforms by promoting
their specific insertion into neuronal membranes.
Given the high degree of homology between the repeat domains of different
ankyrin molecules, we might also expect VGSC- subunits to interact with
other ankyrins such as ankyrinB or ankyrinR. Indeed,
ankyrinR has been shown to bind to uncharacterized native VGSC
preparations from the rat brain
(8). However, it is yet to be
determined whether the affinities for a specific VGSC isoform vary between
different ankyrins.
Data base searches with a consensus sequence for the 9-aa ankyrin-binding
motif found this sequence to be present only in members of the VGSC-
subunit family, suggesting that this particular ankyrin-binding motif is
unique to these proteins. This is in agreement with current models of ankyrin
interactions proposing that different families of ankyrin-binding proteins
independently evolve the ability to bind ankyrin
(16). These searches also
found the motif to be present in VGSC-
subunits from non-mammalian
vertebrates such as the puffer fish, Takifugu pardalis, and the newt.
Intriguingly, this motif was not found in the loop 2
-subunit sequences
of invertebrate sodium channels such as those observed in Drosophila
melanogaster. It would therefore be of some interest to determine whether
or not VGSC/ankyrin-enriched excitable domains can be detected in
Drosophila.
Observations in knockout animals have demonstrated the role of the
cytoskeletal elements ankyrinG and IV spectrin in
establishing and/or maintaining a functional axon IS required for initiation
of an action potential (11,
12,
33). The 9-aa motif described
in this study links VGSCs to these cytoskeletal elements and as such is
necessary to confer the excitable dimension to this domain. The highly
conserved nature of the motif suggests a complex regulation of VGSC/ankyrin
association to achieve isoform specificity but provides a universal code for
the establishment of VGSC-enriched membrane domains through ankyrin
interaction that is likely to be common to a variety of tissues and
organisms.
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FOOTNOTES |
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To whom correspondence should be addressed: Rm. 326, 4 Biotech, 377 Plantation
St., Worcester, MA 01605. Tel.: 508-856-8665; Fax: 508-856-8774; E-mail:
stephen.lambert{at}Umassmed.edu.
1 The abbreviations used are: VGSC, voltage-gated sodium channel; IS, initial
segment; CAM, cell adhesion molecule; MB, membrane binding; GST, glutathione
S-tranferase; aa, amino acid; BHK, baby hamster kidney; -MEM,
minimum essential medium
; HA, hemagglutinin; GFP, green fluorescent
protein; NF, neurofascin; aa, amino acid(s).
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ACKNOWLEDGMENTS |
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REFERENCES |
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