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The Journal of Neuroscience, January 1, 1998, 18(1):128-137
Interaction of Muscle and Brain Sodium Channels with
Multiple Members of the Syntrophin Family
of Dystrophin-Associated Proteins
Stephen H.
Gee1,
Raghavan
Madhavan1,
S. Rock
Levinson2,
John H.
Caldwell2,
Robert
Sealock1, and
Stanley C.
Froehner1
1 Department of Physiology, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7545, and
2 Department of Cellular and Structural Biology, Department
of Physiology and the Neuroscience Program, University of Colorado
Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
Syntrophins are cytoplasmic peripheral membrane proteins of the
dystrophin-associated protein complex (DAPC). Three syntrophin isoforms,  1,  1, and 2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU)
domain, and a PDZ domain. The name PDZ comes from the first three
proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens
protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic
localization of these proteins. Two voltage-gated sodium channels
(NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively,
have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from
extracts of skeletal and cardiac muscle. Peptides corresponding to the
C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind
detergent-solubilized muscle syntrophins, to inhibit the binding of
native NaChs to syntrophin PDZ domain fusion proteins, and to bind
specifically to PDZ domains from 1-, 1-, and 2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the
PDZ domain of neuronal nitric oxide synthase, an interaction that is
not mediated by C-terminal sequences. Brain NaChs, which lack the
(S/T)XV consensus sequence, also copurify with syntrophin and
dystrophin, an interaction that does not appear to be mediated by the
PDZ domain of syntrophin. Collectively, our data suggest that
syntrophins link NaChs to the actin cytoskeleton and the extracellular
matrix via dystrophin and the DAPC.
Key words:
syntrophin; dystrophin complex; PDZ domain; PH domain; neuromuscular junction; sodium channel; cytoskeleton; surface plasmon
resonance
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INTRODUCTION |
To receive and transduce chemical
signals from motor neurons, skeletal muscle cells have developed a
complex, highly organized postsynaptic membrane. The hallmark of this
specialization is the very high concentrations
(~10,000/µm2) of nicotinic acetylcholine
receptors (AChRs) at the crests of the postsynaptic folds (Fertuck and
Salpeter, 1974 ). Voltage-gated sodium channels (NaChs) are also
concentrated in the postsynaptic membrane, where they are primarily
confined to the deeper portions of the folds, and in the perijunctional
membrane (Betz et al., 1984; Beam et al., 1985; Angelides, 1986;
Caldwell et al., 1986; Haimovich et al., 1987; Flucher and Daniels,
1989). This uneven distribution may result in part from a gradient of
NaCh expression by muscle nuclei along the muscle fiber. Locally,
however, it seems most likely to involve NaCh anchoring by cytoskeletal
or other elements (Lupa and Caldwell, 1994).
Compared with the wealth of data on AChR clustering, little is known
about the mechanism of anchoring of NaChs. The synaptic accumulation of
AChRs in skeletal muscle involves the association of AChRs with the
postsynaptic cytoskeleton in response to signals initiated by neurally
derived agrin (McMahan, 1990; Bowe and Fallon, 1995). The 43 kDa
AChR-associated protein rapsyn is believed to play a central role in
this process. Rapsyn clusters AChRs in heterologous cells (Froehner et
al., 1990; Phillips et al., 1991) and in rapsyn-deficient mice, AChRs
are distributed diffusely and do not cluster in response to exogenously
applied agrin (Gautam et al., 1995). Recently, NaChs have been shown to
aggregate at points of contact between skeletal muscle cells and
Chinese hamster ovary (CHO) cells expressing a neural form of agrin,
suggesting that similar mechanisms mediate NaCh anchoring (Sharp and
Caldwell, 1996). However, a cytoskeletal protein with a function
analogous to rapsyn has yet to be identified for NaChs.
The syntrophins are a family of intracellular peripheral membrane
proteins that are components of the dystrophin-associated protein
complex (DAPC) in skeletal muscle. The three known syntrophin isoforms,
1, 1, and 2, are encoded by separate genes and are differentially expressed. All three isoforms are expressed in skeletal
muscle, although 1-syntrophin is the most abundant (Adams et al.,
1993, 1995; Ahn et al., 1994; Yang et al., 1994). Each syntrophin has
two tandem pleckstrin homology (PH) domains followed by a C-terminal
syntrophin-unique (SU) domain (Adams et al., 1995). Inserted in the
first PH domain is a single PDZ domain. The name PDZ comes from the
first three proteins found to contain repeats of this domain (PSD-95,
Drosophila discs large protein, and the zona occludens
protein 1) (Cho et al., 1992). In skeletal muscle, 1- and
1-syntrophin are found on the sarcolemma and are relatively concentrated at the neuromuscular junction (NMJ), whereas
2-syntrophin is concentrated at the NMJ but barely detectable on the
sarcolemma (Peters et al., 1994, 1997). Syntrophins bind directly to
the C-terminal domain of dystrophin and the dystrophin-related proteins utrophin and dystrobrevin (Kramarcy et al., 1994; Ahn and Kunkel, 1995;
Dwyer and Froehner, 1995; Yang et al., 1995; Ahn et al., 1996). The
absence of dystrophin in Duchenne muscular dystrophy (DMD) and the
mdx mouse leads to a dramatic reduction in sarcolemmal syntrophin, although the syntrophins remain concentrated at the NMJ
(Butler et al., 1992; Peters et al. 1994, 1997; Yang et al., 1995).
The PDZ domain of 1-syntrophin is known to bind to the
PDZ-containing N-terminal region of neuronal nitric oxide synthase (nNOS), thereby targeting the enzyme to the sarcolemma (Brenman et al.,
1996a). In other proteins, PDZ domains bind to the C termini of
proteins containing the consensus sequence (S/T)XV-COOH, an interaction
that has been shown to mediate clustering of NMDA receptors and
shaker family K+ channels by the PSD-95
family of synapse-associated proteins (Kim et al., 1995, 1996; Kornau
et al., 1995). We investigated the possibility that syntrophins bind to
ion channels via their PDZ domains. In particular, the subunits of
two skeletal muscle NaChs, SkM1 and SkM2/rH1 (hereafter referred to as
SkM2), have C-terminal sequences with the (S/T)XV motif, and NaChs,
like syntrophins, are present on the sarcolemma and are concentrated at
the NMJ (Froehner et al., 1987; Flucher and Daniels, 1989). We found
that syntrophins, dystrophin, and NaChs can be isolated as stable
complexes from detergent extracts of skeletal and cardiac muscle, and
that the PDZ domains of all three syntrophins bind the C-terminal
sequences of SkM1 and SkM2. Brain NaChs, which lack the C-terminal
(S/T)XV consensus sequence, can also be isolated in a complex with
syntrophins and dystrophin. This interaction does not appear to be
mediated by the PDZ domains of syntrophins. Our data suggest that
syntrophins link NaChs to the actin cytoskeleton and the extracellular
matrix via dystrophin and the DAPC.
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MATERIALS AND METHODS |
Antisera. Affinity-purified polyclonal antibodies to
NaChs were prepared and characterized as described previously
(Dugandzija-Novakovic et al., 1995). In brief, antisera were raised
against a synthetic 18-mer peptide (TEEQKKYYNAMKKLGSKK) corresponding
to a putative intracellular loop connecting homology domains III and
IV, a region that is highly conserved in all functional vertebrate
NaChs. The preparation and characterization of the anti-syntrophin
monoclonal antibody (mAb) 1351 has been described previously (Froehner
et al., 1987). mAb 1351 is directed against an epitope within the PDZ
domain of syntrophin (S. H. Gee and S. C. Froehner,
unpublished data) and recognizes all known mouse syntrophin isoforms
(Peters et al., 1994, 1997). mAb 1808 against dystrophin has been
described previously (Sealock et al., 1991). Isoform-specific
monoclonal antibodies to K+ channel subunits
(Bekele-Arcuri et al., 1996) were a generous gift from Dr. James S. Trimmer (State University of New York at Stony Brook).
Fusion proteins. Mouse 1-syntrophin domains were
generated by PCR amplification of an 1-syntrophin cDNA clone
(GenBank accession number U00677) with oligonucleotide primers flanking
nucleotides 111-341 (PH1a), 333-620 (PDZ), 594-923 (PH1b), 948-1328
(PH2), and 1323-1622 (SU) (Adams et al., 1993; Brenman et al., 1996a). For the 2-PDZ domain, the primers flanked nucleotides 320-594 (corresponding to amino acids 90-184) of a cDNA encoding mouse 2-syntrophin (GenBank accession number U00678) (Adams et al., 1993). For the 1-PDZ domain, the primers flanked nucleotides 667-954 (corresponding to amino acids 115-210) of a cDNA encoding mouse 1-syntrophin (GenBank accession number U89997) (Peters et al.
1997). The resultant products were subcloned into the pET-32a vector
(Novagen, Madison, WI) that contains sequences encoding thioredoxin, an
N-terminal 15 amino acid S-Tag, and a hexahistidine nickel binding
motif. The 3 end of the PH1a PCR product was ligated to the 5 end of
the PH1b PCR product (using engineered XbaI sites) to
produce an intact PH1 domain. Positive clones were selected and
sequenced. Clones with the correct sequence were electroporated into
BL21( DE3)pLysS competent cells. Overnight cultures were diluted
1:10, grown at 37°C for 2 hr, and then induced for 17 hr at 28°C
with 1 mM
isopropyl--D-thiogalactopyranoside. The expressed fusion
proteins were all recognized by the S-Protein HRP conjugate (Novagen)
and were purified on nickel-Sepharose columns according to the
manufacturer's instructions. All domains were purified from the
soluble fraction. Fusion protein purity was determined by Coomassie
blue staining of SDS-polyacrylamide gels. Protein concentration was
determined by the method of Bradford (1976). Fusion proteins linking
N-terminal domains of nNOS (amino acids 1-150 and 1-299) and
glutathione S-transferase (GST) were generated as described
(Brenman et al., 1996a,b).
Peptides. Synthetic peptides corresponding to the C-terminal
10 amino acids of the adult skeletal muscle NaCh (SkM1; VRPGVKESLV), the cardiac and embryonic skeletal muscle NaCh (SkM2; SPDRDRESIV), and
the NMDA receptor 2B subunit (NR2B; EKLSSIESDV) were purchased from
Macromolecular Resources (Colorado State University, Fort Collins, CO).
Fas (NFRNEIQLSLV) and adenomatous polyposis coli (APC; HSGSYLVTSV)
peptides were a generous gift from Dr. Brian Kay and Stacy Sekely
(University of North Carolina at Chapel Hill). All peptides contained
an additional four amino acid linker (SGSG) at the N terminus and an
N-terminal biotin.
Preparation of detergent-solubilized membrane extracts.
Tissues were dissected from C57BL6 mice or Sprague Dawley rats and were
frozen in liquid nitrogen or used fresh as needed. Tissue was
homogenized in a Waring blender in 10 vol of 50 mM Tris, pH 7.5, 1 mM EDTA, and 1 mM EGTA (TEE). The
following protease inhibitors were added immediately before
homogenization: 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml each leupeptin, pepstatin, aprotinin, and antipain. The
homogenate was centrifuged at 30,000 × g for 10 min,
and the supernatant was removed. The pellet was rehomogenized in 10 vol
of the same buffer and then centrifuged as above. The final pellet was
resuspended in 10 vol of TEE plus 100 mM NaCl plus 1%
Triton X-100, extracted for 30 min at 4°C on a rocker platform, and
then clarified by centrifuging at 40,000 × g for 20 min. The supernatant was removed from beneath the fat layer, if
present.
Peptide affinity chromatography. Biotinylated SkM1, SkM2,
and NR2B peptides (200 µg each) were coupled to 0.5 ml of
streptavidin-agarose (Sigma, St. Louis, MO) in PBS, pH 7.2, for 5 hr
at 4°C on a rocker platform. The beads were then washed extensively
with TEE plus 350 mM NaCl plus 0.1% Triton X-100 to remove
unbound peptide. Two hundred microliters of a 50% slurry of each
agarose-coupled peptide or of uncoupled streptavidin-agarose beads
were added to 1 ml of detergent-solubilized cardiac muscle membranes
and incubated overnight at 4°C on a rocker platform. After extensive washing with the above buffer, the beads were aspirated to near dryness
and heated to 80°C in 100 µl of SDS-PAGE loading buffer.
Immunoaffinity purification. mAb 1351 against syntrophin,
mAb 1808 against dystrophin, and control mouse IgG (4 mg each) were coupled to Affi-Gel 10 (Bio-Rad, Hercules, CA) in 20 mM
HEPES, pH 7.5, plus 150 mM NaCl according to the
manufacturer's instructions. These antibody resins were incubated with
10 ml of detergent-solubilized membranes for 1 hr at 4°C and then
were washed extensively with TEE plus 0.5 M NaCl plus 1%
Triton X-100, followed by washing with TEE plus 0.5 M NaCl
plus 0.1% Triton X-100 and finally with TEE. Bound proteins were
eluted with 2.75 ml of 0.1 M triethylamine, pH 11.5, and
were immediately neutralized by the addition of 1.25 ml of 1.5 M Tris, pH 6.8. Eluted proteins were precipitated by the
addition of 0.5 ml 100% trichloroacetic acid (TCA; final
concentration, 12%) and overnight incubation on ice. Proteins were
pelleted by centrifugation at 39,000 × g for 1 hr in
an SW41Ti rotor (Beckman Instruments) after addition of 12% TCA in TEE
to fill the centrifuge tubes. The pellets were rinsed with 99%
ethanol, dried, and then resuspended in SDS-PAGE loading buffer.
Fusion protein affinity chromatograpy. Solubilized membranes
from mouse cardiac muscle or brain were prepared as described above.
Fifty micrograms of purified syntrophin fusion protein were added to 1 ml of extract in 1.4 ml Eppendorf tubes along with 25 µl of
S-Protein-agarose beads and incubated overnight at 4°C on a rocker
platform. After centrifugation, the S-Protein beads were washed
extensively with TEE plus 0.5 M NaCl plus 1% Triton X-100.
The final wash was removed completely by aspirating with a flat
gel-loading tip. The beads were resuspended in 50 µl of SDS-PAGE
loading buffer and heated at ~80°C for 5 min. For peptide
competition experiments, syntrophin PDZ domain fusion proteins were
preincubated with 10 µM peptide for 30 min on ice.
Immunoblotting and overlay assays. Proteins were
resolved by SDS-PAGE and transferred onto nitrocellulose membranes
(Towbin et al., 1979). The membranes were blocked with 5% skim milk
powder in 25 mM Tris, pH 7.5, 150 mM NaCl, and
0.1% Tween 20 (TBS-Tween), and then incubated with primary antibody
in the same buffer for 1 hr at room temperature. The blots were washed
three times for 10 min each in TBS-Tween, incubated with
HRP-conjugated secondary antibody for 30 min, and finally washed
another four times for 10 min each. Immunoreactive bands were
visualized by enhanced chemiluminescence. Overlays with biotinylated
synthetic peptides were carried out as above with the following
modifications. Biotinylated peptides were preconjugated to
streptavidin-HRP as follows. Streptavidin-HRP (0.66 µg/ml) was
added to 0.1 µM peptide in blocking buffer and incubated
for 30 min at room temperature on a rocker platform. Blots were
incubated with the peptide conjugates for 1 hr and then washed four
times for 15 min each with TBS-Tween. Overlays with nNOS were carried
out as described previously (Brenman et al., 1996a). The SkM1 and NR2B
peptides were added where indicated to a final concentration of 1 µM.
Surface plasmon resonance. The relative binding of
syntrophin PDZ domains to SKM1, SKM2, NR2B, APC, and Fas peptides was
determined using surface plasmon resonance (Morelock et al., 1995;
Myszka et al., 1996). This technique has been discussed in several
reviews (Fisher and Fivash, 1994; O'Shannessy, 1994; Myszka, 1997). In brief, ligands are attached to a carboxymethylated dextran gold surface, and analytes are injected over the peptide surface. Binding is
measured as an increase in resonance units (RU), which directly reflect
changes in refractive index at the surface of the chip. All experiments
were performed on a BIAcore 2000 instrument at the University of North
Carolina Macromolecular Interactions Facility. Neutravidin (Pierce,
Rockford, IL)-coated CM5 sensor chips (BIAcore, Piscataway, NJ) were
prepared to capture the biotinylated peptides onto the surface.
Neutravidin was dissolved at 1 mg/ml in 20 mM HEPES, pH
7.4, 3.4 mM EDTA, 150 mM NaCl, and 0.005%
surfactant P-20 (BIAcore) (HBS). The neutravidin solution was diluted
1:20 in 10 mM sodium acetate, pH 6.0, and then covalently
attached to the carboxyl groups on the surface of the CM5 sensor chip
activated with 200 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 50 mM N-hydroxysuccinimide. This was followed by 10 injections of 100 mM HCl (2 min contact time) to achieve a
stable baseline. Biotinylated NaCh peptides were immobilized by one or
two injections of 100 nM peptide onto the neutravidin
surface. After injection of 10 mM HCl to remove unbound
peptide, final surface densities varied between 75 and 125 RU.
To measure relative binding, purified syntrophin PDZ domain fusion
proteins in HBS were injected onto the peptide surfaces at a flow rate
of 20 µl/min for 2 min. The steady-state binding levels, in RU, were
recorded at the end of each injection. Between successive measurements
the surfaces were regenerated by treatment with 10 mM HCl
(2 min contact time). Binding to a control blank flow cell was also
measured and used to subtract nonspecific interactions. To normalize
the relative responses to the amount of immobilized peptide, the RU
values recorded at the end of the sensorgrams were divided by the
amount of each specific peptide immobilized.
To study the inhibition of binding of 1-syntrophin PDZ domain to
nNOS by peptides, a monoclonal antibody to GST (Santa Cruz Biotechnology, Santa Cruz, CA) was covalently coupled to a CM5 sensor
chip as above. The GST mAb solution was diluted to 5 µg/ml in 10 mM sodium acetate, pH 5.75, and then injected at a flow rate of 20 µl/min for 2 min. After injection of 100 mM
NaOH (5 min contact time) to remove unbound antibody, final surface
densities varied between 3000 and 10,000 RU. A 100 µg/ml solution (in
HBS) of a GST fusion protein encoding the first 299 amino acids of nNOS
(Brenman et al., 1996a) was injected onto the chip surface (1 min at a
flow rate of 20 µl/min) followed by injection of a 1 µM
solution of 1-syntrophin PDZ domain fusion protein (2 min contact
time). Peptides were mixed with the 1-PDZ solution just before
injection. Between successive measurements the surface of the chip was
regenerated by treatment with 3 M MgCl2 (5 min contact time).
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RESULTS |
Voltage-gated NaChs are composed of a single transmembrane,
channel-forming subunit (Mr ~250 kDa) and
a variable number of smaller (Mr ~35 kDa)
transmembrane auxiliary subunits. At least 11 different, but
homologous, genes encode the subunits, each of which is composed of
four domains having six transmembrane helices each. The subunits
expressed in adult skeletal muscle (designated SkM1) and in fetal
skeletal muscle and cardiac muscle (SkM2) have similar C termini that
contain the consensus sequence (Q/E)(S/T)XV-COOH predicted to be a
ligand for syntrophin PDZ domains (Songyang et al., 1997).
Muscle and brain NaChs copurify with syntrophins
and dystrophin
We first determined whether muscle NaChs exist in a complex with
syntrophin and dystrophin. Detergent-solubilized membrane extracts of
mouse skeletal muscle were incubated with the pan-specific, anti-syntrophin antibody mAb 1351 coupled to Affi-Gel-10 agarose. After
extensive washing with buffer containing 0.5 M NaCl and 1%
Triton X-100, bound proteins were eluted with pH 11.5 buffer, neutralized, and resolved by SDS-PAGE. Immunoblotting of these syntrophin preparations with a polyclonal antibody to a region conserved in all known NaCh subunits revealed a band of ~250 kDa,
consistent with the size of intact NaCh subunit (Fig.
1A, NaCh).
Preabsorption of the NaCh antibody with the immunizing peptide
eliminated this immunoreactivity (Fig. 1A,
+pept). As expected, these preparations also contained
full-length dystrophin (Kramarcy et al., 1994) and smaller bands, which
likely correspond to shorter dystrophin isoforms (Ahn and Kunkel, 1993)
(Fig. 1A, Dys). Eluates from control mouse
IgG-agarose contained no detectable syntrophin, dystrophin, or NaCh.
In the reciprocal experiment, immunoaffinity preparations of dystrophin
contained both syntrophin and NaCh (Fig. 1B).
Collectively, these data suggest that one or more syntrophins,
dystrophin, and NaCh exist as a stable complex in detergent extracts of
skeletal muscle and, presumably also, in situ. Similar
results were also obtained with extracts of mouse cardiac muscle and
with rat skeletal and cardiac muscle (data not shown).
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Figure 1.
Syntrophin, dystrophin and NaChs form a stable
complex in skeletal muscle and brain. Detergent-solubilized mouse
skeletal muscle or brain membranes were incubated with Affi-Gel-10
agarose coupled to control mouse IgG (IgG), a
pan-specific mAb to syntrophins (Syn), or an mAb to
dystrophin (Dys). Immunoaffinity-purified complexes were
analyzed by immunoblotting with mAbs specific for syntrophin and
dystrophin or with a polyclonal antibody to NaChs. Immunoblots for NaCh
were performed in the absence (NaCh), or presence
(+pept) of excess immunizing peptide. A,
Immunoaffinity-purified syntrophin preparations contain full-length
dystrophin (and two shorter forms or proteolytic products of
dystrophin) and NaCh. B, Immunoaffinity-purified
dystrophin complexes contain both syntrophin and NaCh.
C, Syntrophin preparations from brain contain dystrophin and NaCh. The positions of molecular mass markers (in kilodaltons) are
shown at the left.
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Because none of the known brain NaChs contain the consensus C-terminal
sequence for binding to syntrophin PDZ domains, we also purified
syntrophins from mouse brain as a control, in the expectation that
these preparations would not contain NaChs. In fact, they also
contained NaChs as well as dystrophin (Fig. 1C). These data
suggest that syntrophins may also exist in a stable complex with brain
NaChs, presumably through interactions that do not involve the PDZ
domain.
The PDZ domain of syntrophin binds muscle, but not
brain, NaChs
To determine which domains of syntrophin are necessary for binding
to NaChs, we generated fusion proteins linking thioredoxin and a 15 amino acid epitope tag (S-Tag) to individual protein domains of
1-syntrophin (Fig.
2A). The S-Tag permits
recovery and detection of fusion proteins via its interaction with
S-Protein. Approximately equal amounts of each fusion protein were
incubated with Triton X-100 extracts of mouse cardiac muscle or brain
and then isolated with S-Protein-agarose. On immunoblots of the
eluates, bound fusion proteins were detected using HRP-conjugated
S-Protein. Approximately equal amounts of fusion protein were captured
in each case (data not shown). As expected, cardiac muscle NaCh was captured only by the PDZ domain fusion protein (Fig.
2B, left). Amounts captured by other
syntrophin domains were negligible. In several additional experiments
these amounts were comparable to that captured by thioredoxin alone
(data not shown). No NaCh was detected in eluates from
S-Protein-agarose beads alone.
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Figure 2.
The PDZ domain of 1-syntrophin captures
NaChs from muscle but not brain. A, Domain organization
of 1-syntrophin and position of corresponding fusion protein
constructs. 1-Syntrophin domains corresponding to those described by
Adams et al. (1995) are shown drawn to scale; the first and last amino
acids in each domain are numbered. Represented
below are thioredoxin fusion proteins of individual
syntrophin domains containing a 15 amino acid epitope tag (S-Tag) that
binds to S-Protein. B, Affinity isolation of NaChs from
mouse heart and brain with 1-syntrophin domain fusion proteins. The
indicated 1-syntrophin fusion proteins (PH1,
PDZ, PH2, and SU)
were incubated with detergent-solubilized membranes of mouse heart or
brain, with (+) or without ( ) 10 µM SkM2 peptide, and
S-Protein-agarose beads. Approximately equal amounts of each fusion
protein were precipitated by the S-Protein-agarose beads, as determined
by staining with Ponceau S and by blotting with S-Protein conjugated to
HRP (data not shown). The PDZ domain of 1-syntrophin captured NaCh
from heart but not from brain. Capture of NaCh from heart was inhibited
completely by addition of SkM2 peptide. No NaCh was captured by
thioredoxin alone (Trx) or by S-Protein-agarose beads
alone (S).
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Immunoblotting of the eluates from brain (Fig. 2B,
right) revealed that the fusion protein containing the
1-syntrophin PDZ domain failed to capture NaChs, as expected. The
other three fusion proteins did, however, capture NaChs. In several
experiments the SU domain consistently bound more NaCh than either the
PH1 or PH2 domains (Fig. 2B, right).
Collectively, our results suggest that the syntrophin PDZ domain binds
specifically to the C-terminal SXV motif of muscle NaChs, whereas other
syntrophin domains contribute to the binding of brain NaChs.
The C terminus of muscle NaChs binds to the PDZ domain
of syntrophins
We next determined whether the C-terminal peptide binding activity
of PDZ domains (Doyle et al., 1996) could account for interaction between SkM2 and syntrophin PDZ domains. Preincubation of syntrophin domain fusion protein with a peptide corresponding to the SkM2 NaCh C
terminus completely blocked the ability of the PDZ domain to capture
NaCh from cardiac extracts (Fig. 2B, left,
lanes marked ±).
To determine whether the PDZ domains of all three syntrophins are
capable of binding NaChs with a C-terminal SXV motif, PDZ domain fusion
proteins were used to isolate NaChs from heart extracts. As shown in
Figure 3A, all three captured
NaChs from cardiac muscle, although 1-PDZ bound significantly less
than either 1- or 2-PDZ. No NaCh was captured by
S-Protein-agarose beads alone. In a separate experiment, syntrophin
PDZ domain fusion proteins were preincubated with a non-SXV control
peptide or a peptide corresponding to the SkM2 NaCh C terminus and then
used to capture NaChs from cardiac muscle extracts. In the presence of
control peptide (Fig. 3B, lanes C), all three
syntrophin PDZ domains bound NaCh at levels comparable to those in the
absence of peptide (Fig. 3A). The 1-PDZ domain, again,
bound much less NaCh than the other two. As found previously for
1-PDZ (Fig. 2B, left), preincubation
with the NaCh C-terminal peptide inhibited capture of NaCh by all three fusion proteins (Fig. 3B, lanes Na).
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Figure 3.
The C terminus is necessary for the interaction of
the cardiac muscle NaCh with the PDZ domains of 1, 1, and
2-syntrophin. A, Affinity isolation of NaChs from
heart with syntrophin PDZ domains. Detergent-solubilized mouse heart
membranes were incubated with 1-, 1-, or 2-syntrophin PDZ
domains and S-Protein-agarose. As in Figure 2, S-Protein-agarose
beads pulled down approximately equal amounts of fusion protein (data
not shown). 1- and 2-PDZ pulled down roughly equal amounts of
NaCh, whereas 1-PDZ pulled down substantially less.
B, The SkM2 NaCh C-terminal peptide inhibits binding to
syntrophin PDZ domains. Syntrophin PDZ domain fusion proteins were
added to cardiac muscle membrane extracts (as in A),
except that PDZ fusion proteins were preincubated with 1 µM non-SXV control peptide (lanes C) or a
peptide corresponding to the C-terminal 10 amino acids of the SkM2 NaCh
(lanes Na). The SkM2 peptide almost completely blocks the
interaction of cardiac NaChs with syntrophin PDZ domains. As in
A, 1- and 2-PDZ pulled down roughly equal amounts
of NaCh, whereas 1-PDZ pulled down substantially less.
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To determine whether the C terminus alone of NaChs is sufficient for
binding to syntrophins, biotinylated peptides corresponding to the
C-terminal sequences of SkM1 (VRPGVKESLV), SkM2 (SPDRDRESIV), and the
NMDA receptor 2B subunit (NR2B; EKLSSIESDV) were coupled to
streptavidin-agarose and used to isolate syntrophins from cardiac muscle extracts. Figure 4 shows that the
SkM1 and SkM2 peptides bound syntrophins well, with the SkM2 peptide
binding more syntrophin than the SkM1 peptide. In this assay, the NR2B
peptide, which also corresponds to the consensus sequence
(Q/E)(S/T)XV-COOH for syntrophin PDZ binding, bound very small amounts
of syntrophin. Streptavidin-agarose alone did not bind syntrophins
detectably.
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Figure 4.
The C termini of muscle NaChs are sufficient for
interaction with syntrophins. Approximately equal amounts of
biotinylated peptides corresponding to the C-terminal 10 amino acids of
the adult skeletal (SkM1) and cardiac
(SkM2) muscle NaChs and the NMDA receptor 2B subunit
(NR2B) were coupled to streptavidin-agarose beads.
Detergent-solubilized heart membranes were incubated with peptide-conjugated beads or with beads alone (). Bound proteins were
immunoblotted for syntrophin. Both the SkM1 and SkM2 peptides bound
substantial amounts of syntrophin, whereas NR2B bound much less.
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The results described above do not exclude the possibility that an
additional protein mediates the interaction between the NaCh C termini
and syntrophin PDZ domains. To determine whether the interaction is
direct, the biotinylated SkM1 and SkM2 peptides were coupled to
streptavidin-HRP and used to overlay protein blots of 1-syntrophin
domain fusion proteins (Fig.
5A). A significant fraction of
all three syntrophin PDZ domain fusion proteins consistently migrated
in SDS-polyacrylamide gels as an ~60 kDa band, approximately twice
the size expected. The basis for this apparent dimerization is not
known. Both peptides bound to the PDZ domain but not to the PH1, PH2,
or SU domains. Notably, more SkM2 peptide bound to the 1-PDZ domain
than did the SkM1 peptide. A peptide corresponding to the C terminus of
Fas (NFRNEIQLSLV) (Sato et al., 1995) did not bind to any of the
domains. The NaCh peptide sequences also appear to be specific for
syntrophin PDZ domains. The SkM1 and SkM2 peptides, but not the Fas
peptide, bound directly to the PDZ domains of all three syntrophins
(Fig. 5B) but did not bind to the PDZ domain of nNOS. These
data and the failure of brain NaChs to bind to syntrophin PDZ domains
suggest that the C-terminal sequences of muscle NaCh subunits are
both necessary and sufficient for binding to syntrophin PDZ
domains.
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Figure 5.
The C termini of muscle NaChs bind directly to
syntrophin PDZ domains. A, Biotinylated peptides
corresponding to the C-terminal 10 amino acids of the SkM1 and SkM2
NaChs or Fas bound to streptavidin-HRP were overlaid onto
1-syntrophin domain fusion proteins. The position and relative
amount of each fusion protein was determined by blotting with
S-Protein-HRP. The asterisk indicates the position of
the PH1 fusion protein. Both the SkM1 and SkM2 peptides bind
exclusively to the PDZ domain of 1-syntrophin, whereas the Fas
peptide did not bind to any of the domains. B, SkM1,
SkM2, and Fas C-terminal peptides were overlaid onto 1-, 1-, and
2-syntrophin PDZ domain fusion proteins and a GST fusion protein of
nNOS (amino acids 1-150) containing the PDZ domain
(NOS). The position and relative amount of each fusion
protein were determined by blotting with S-Protein-HRP or with an mAb
to GST (Anti-GST), followed by donkey anti-mouse-HRP (DAM-HRP). A significant fraction of all
three syntrophin PDZ domain fusion proteins migrated as dimers. SkM1 and SkM2 peptides bound to the PDZ domains of all three syntrophins but
not to the PDZ domain of nNOS. In contrast, the Fas peptide did not
bind to any of the PDZ domains.
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Syntrophin PDZ domains bind preferentially to SkM2 NaCh
C-terminal peptides
Relative strengths of the interactions between C-terminal S/TXV
sequences and syntrophin PDZ domains were measured using surface plasmon resonance. The three syntrophin PDZ domains were tested against
biotinylated C-terminal peptides from the following proteins: SkM1,
SkM2, NR2B, Fas, and APC (HSGSYLVTSV). As shown in Figure 6, all three syntrophin PDZs bound best
to the SkM2 NaCh peptide. Each also bound the SkM1 NaCh peptide and,
interestingly, the NR2B peptide, albeit to a lesser extent than to the
SkM2 peptide. In contrast, binding to both Fas and APC peptides was
minimal, suggesting that amino acids upstream of S/TXV may also
influence binding of these C-terminal peptides to syntrophin PDZ
domains.
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Figure 6.
Syntrophin PDZ domains bind to specific C-terminal
SXV peptides. The ability of syntrophin PDZ domain fusion proteins to
bind to SkM1, SkM2, NR2B, Fas, and APC peptides was quantitated by surface plasmon resonance. Data for the binding of 500 nM
1-PDZ (black bars), 1-PDZ (gray
bars), and 2-PDZ (open bars) are taken from a
single time point in the steady-state part of the binding reaction and
are the average of two separate determinations. The relative response
(resonance units, RU) corresponds to the amount of fusion protein bound and was normalized to the amount of peptide immobilized in each flow cell. Error bars represent SEM.
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To address further the role of sequences upstream of S/TXV, we used
pull-down assays to determine whether syntrophin PDZ domain fusion
proteins could interact with three members of the shaker family of K+ channels: Kv1.4 (VETDV), Kv1.1 (LLTDV),
and Kv1.2 (MLTDV). Kv1.4 has been shown previously to bind to the
second PDZ domain of PSD-95 (Kim et al., 1995). We found that all three
syntrophin PDZ domains captured substantial amounts of Kv1.4 from
detergent extracts of mouse brain membranes but very little of either
Kv1.1 or Kv1.2, as detected by immunoblotting (data not shown). These results suggest that an E at the 3 position from the C terminus of
the S/TXV peptide is important for strong syntrophin PDZ domain interaction. These results also show that syntrophin PDZ domains can
interact with sequences with either S or T at the 2 position. A
summary of the syntrophin PDZ domain sequence preferences, based on
these findings, is presented in Figure
7.
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Figure 7.
Summary of the binding of syntrophins to
C-terminal SXV sequences. Residues in common with those in SkM1 or SkM2
are shaded. The consensus sequence for binding to
syntrophin PDZ domains is shown below the
line, with residues important for strongest binding to
syntrophin PDZ domains in bold. The methods used to
conclude whether a given sequence binds to syntrophin PDZ domains are
indicated at the right. a, Immunoaffinity
copurification; b, affinity isolation with PDZ fusion
proteins; c, peptide overlay assays; d,
surface plasmon resonance; e, peptide affinity
chromatography. RBSC, Rat brain sodium channel.
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K+ channels bind to syntrophin PDZ domains but
do not copurify with syntrophins
Because syntrophin PDZs can interact with shaker
K+ channels (especially Kv1.4; see above), we asked
whether these proteins are present in syntrophin preparations. In
contrast to our results with NaChs, we were unable to detect
K+ channels in syntrophin preparations from brain,
even though we could readily detect them in the membrane extracts (data
not shown). Thus, if K+ channels exist in complexes
with syntrophins in situ, the complexes may be of such low
affinity that they dissociate after detergent solubilization. It is
noteworthy that although syntrophin PDZ-K+ channel
complexes did not form in the extracts under our conditions of
immunopurification, they could form under pulldown conditions (i.e., a
large excess of PDZ fusion protein added to the extracts). From this,
we conclude that the presence of NaChs in syntrophin preparations
reflects specific complexes that exist in situ, before detergent solubilization.
The C-terminal peptide of SkM2 inhibits binding of the
1-syntrophin PDZ domain to nNOS
The PDZ domain of 1-syntrophin and the second PDZ domain of
PSD-95 bind directly to nNOS fusion proteins containing a PDZ domain,
probably by forming PDZ-PDZ heterodimers (Brenman et al., 1996a).
Because the PDZ domain of nNOS is internal to the protein, the
interaction cannot be via C-terminal binding. The interaction of nNOS
and PSD-95 is inhibited by the C-terminal peptide of NR2B (Brenman et
al., 1996a), which binds strongly to the second PDZ domain of PSD-95
(Kornau et al., 1995). This suggests that the two binding reactions
involve overlapping sites or that there is strong allosteric coupling
between sites. To determine whether syntrophin PDZ domains exhibit this
property, we tested NaCh C-terminal peptides for inhibition of the
binding of the 1-syntrophin PDZ domain to an nNOS PDZ-containing
fusion protein (GST-nNOS 1-299) (Brenman et al., 1996a). First, we
tested whether an NaCh C-terminal peptide could inhibit the binding of
nNOS fusion protein to 1-PDZ in overlay assays. As shown in Figure
8A, SkM1 peptide (1 µM) completely inhibited the binding of nNOS to 1-PDZ.
In contrast, addition of the same concentration of either non-SXV
peptides or NR2B had no effect. We also tested the ability of the SkM1 peptide to inhibit 1-PDZ binding to nNOS using surface plasmon resonance. GST-nNOS (1-299) fusion protein was immobilized on a sensor
chip, and 1-PDZ was injected onto the surface of the chip. In the
absence of added peptide, injection of 1-PDZ resulted in a large
increase in the relative response, indicative of 1-PDZ binding to
the nNOS fusion protein (Fig. 8B, top
trace). Addition of 1 µM peptide decreased the
observed response to approximately half of the control response (Fig.
8B, middle trace). The response was
further reduced when 10 µM peptide was added (Fig.
8B, bottom trace). These data demonstrate
that NaChs and nNOS bind to the same or overlapping sites on the PDZ
domain of 1-syntrophin. Thus, it appears unlikely that nNOS and
NaChs bind simultaneously to syntrophin PDZ domains.
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Figure 8.
Competition of nNOS binding to 1-syntrophin PDZ
domain by the SkM1 NaCh C-terminal peptide. A, Purified
1-syntrophin PDZ domain fusion protein was resolved by SDS-PAGE and
transferred to a nitrocellulose membrane. Individual
lanes were incubated with a GST-nNOS (1-299) fusion
protein either alone () or in the presence of 1 µM
control non-SXV peptides (Control 1, 2), NR2B C-terminal
peptide (NR2B), or SkM1 C-terminal peptide
(SkM1). nNOS bound to both the monomer and dimer of
1-PDZ. Binding was almost completely blocked by the SkM1 peptide.
B, Surface plasmon resonance. GST-nNOS (1-299) fusion
protein was injected onto the surface of a sensor chip coated with an
mAb to GST, followed by injection of purified 1-syntrophin PDZ
domain fusion protein in the absence (top trace) or
presence of the indicated concentrations of SkM1 C-terminal peptide
(middle and bottom traces). Binding of
1-PDZ to GST-nNOS was measured in real time as an increase in
resonance units (RU). One micromolar SkM1 peptide
reduced the binding by approximately half, and 10 µM
peptide almost completely inhibited the binding of 1-PDZ to nNOS
(the remaining response is attributable primarily to bulk changes in
refractive index).
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DISCUSSION |
We have shown that syntrophins bind to adult skeletal (SkM1) and
cardiac (SkM2) muscle NaChs. This interaction is mediated by direct
binding of the PDZ domains of syntrophins to the (S/T)XV C terminus of
these NaChs and may link NaChs to the extracellular matrix and the
cortical actin network. Formation of this complex (Fig.
9) may be relevant to the proper
localization and physiological function of NaChs in muscle.
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Figure 9.
Model of NaCh interactions with the DAPC in
muscle. Syntrophin (SYN) is shown bound to the
C-terminal domain of dystrophin (DYS). Dystrophin
associates directly with -dystroglycan (-DG), which in turn binds -dystroglycan (-DG). The PDZ
domain of syntrophin (rectangle) is bound to the
C-terminal SXV sequence of a NaCh. SG, Sarcoglycan
complex.
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The C termini of the SkM1 and SkM2 NaChs are sufficient to bind
detergent-solubilized syntrophins from muscle, to inhibit the binding
of NaChs to syntrophin PDZ domain fusion proteins, and to bind directly
to syntrophin PDZ domains. To a lesser extent, syntrophin PDZ domains
also interact with Kv1.4 channels and the NMDA receptor 2B subunit.
Binding to Fas or APC C-terminal peptides was negligible. Thus,
syntrophin PDZ domains exhibit a high degree of specificity for
C-terminal peptides (see Fig. 7).
These results can be rationalized on the basis of the crystal structure
of a PDZ domain complexed with its cognate ligand (Doyle et al., 1996).
In the third PDZ domain of PSD-95, a loop formed by the conserved GLGF
sequence forms a hydrophobic pocket which binds the C-terminal Val (0 position) of the peptide. Side chains of residues at positions 2 and
3 make contacts with side chains in the peptide-binding pocket of the
PDZ domain, whereas the side chain of the residue at position 1
points away from the binding pocket and therefore is not predicted to
influence binding. Our studies clearly demonstrate that C-terminal SXV
sequences are important for binding syntrophin PDZ domains (Fig. 7).
For example, brain NaChs do not bind to syntrophin PDZ domains but have
C-terminal sequences (especially RBSC II and RBSC VI) that are highly
similar to muscle NaChs, except that they lack a Val at the 0 position.
The residue at the 3 position also appears to be essential for
binding to syntrophin PDZ domains, because all of the peptides that
bound had a Glu at this position. For example, the last three amino
acids of Fas are identical to SkM1, yet the Fas peptide, which has a
Gln at the 3 position, did not bind to the PDZ domain of any of the
syntrophins. Finally, the residue at position 4 may be important for
binding of peptides to syntrophin PDZ domains. The SkM2 NaCh peptide,
which has an Arg at 4, bound approximately fourfold better (as
measured by the relative response; Fig. 6) to syntrophin PDZs than
either the SkM1 or NR2B peptides, which have a Lys or Ile,
respectively, at this position. It has been suggested that a Lys at
position 4 can bind a Glu in the loop between the B and C
strands of the PDZ domain (Songyang et al., 1997), a residue conserved
in the syntrophin PDZ domain. Perhaps then, an Arg at the 4 position confers stronger binding than a corresponding Lys. Interestingly, syntrophins contain GLGI in place of the well conserved GLGF sequence. Our results demonstrate that PDZ domains containing GLGI can also bind
C-terminal peptides with an SXV consensus sequence.
A surprising result of our studies was the finding that brain NaChs,
which lack the SXV C-terminal consensus sequence, also copurify with
syntrophins. As expected, the PDZ domain of 1-syntrophin did not
bind any brain NaChs but did bind to the SXV-containing muscle NaChs.
We did find, however, that NaChs from brain were captured by the SU and
PH1 domains, and to a lesser extent by the PH2 domain, of
1-syntrophin. The SU domain consistently captured more NaCh than
either the PH1 or PH2 domains. Collectively, our results suggest that
other domains in syntrophin can contribute to binding brain NaChs.
If the interaction of the PH and SU domains of syntrophin with brain
NaChs is direct, it may occur via binding to other intracellular domains of NaChs. Alternatively, other proteins associated with NaChs
may mediate this interaction. The two unrelated auxiliary subunits
of NaChs, designated 1 and 2, are possibilities (Isom et al.,
1992, 1995). Another possibility is ankyrin, which copurifies with
NaChs from brain (Srinivasan et al., 1988). In skeletal muscle, ankyrin
is colocalized with NaChs in the troughs of the postjunctional folds
(Flucher and Daniels, 1989); in brain, ankyrin is colocalized with
NaChs at the nodes of Ranvier on myelinated axons (Kordeli et al.,
1990). In any case, our results suggest that NaChs in brain, like those
in muscle, may be associated with the dystrophin or related
proteins.
What is the functional significance of the association of NaChs with
syntrophin and dystrophin? The ability of syntrophin to link NaChs to
dystrophin and the DAPC is reminiscent of the AChR-associated protein
rapsyn. Cotransfection experiments have demonstrated that rapsyn can
cluster AChRs (Froehner et al., 1990; Phillips et al., 1991; Apel et
al., 1995) and - and -dystroglycan (Apel et al., 1995, 1997),
extracellular and transmembrane proteins, respectively, of the DAPC
(Ervasti and Campbell, 1991). Thus, rapsyn may function as a link
between AChRs and the DAPC. Syntrophins may play an analogous role in
the anchoring or clustering of NaChs. Because dystrophin binds actin
(Hemmings et al., 1992), and -dystroglycan binds both laminin
(Ibraghimov-Beskrovnaya et al., 1992; Ervasti and Campbell, 1993; Gee
et al., 1993) and agrin (Bowe et al., 1994; Campanelli et al., 1994;
Gee et al., 1994; Sugiyama et al., 1994), the interaction with
syntrophins may be sufficient to link NaChs to the cortical actin
network and the extracellular matrix. Consistent with this idea, CHO
cells expressing a neural agrin isoform can cluster NaChs at points of
contact with muscle cells (Sharp and Caldwell, 1996).
Finally, syntrophins may modulate the function of NaChs. Evidence
supporting such a notion is the recent demonstration that the
interaction of the SXV-containing C terminus of the
K+ channel subunit Kv1.1 with the cytoskeleton
regulates the extent of inactivation conferred by the subunit (Jing
et al., 1997). Such functional changes may be especially important when
considering myopathies such as DMD. For example, patients with DMD
suffer from conduction disturbances and heart block, suggesting an
important role for dystrophin in the cardiac conduction system (Bies et al., 1992). The finding that syntrophin links NaChs to dystrophin provides a plausible explanation for these defects; a reduction in
sarcolemmal syntrophin may lead to aberrant NaCh distribution or
function in cardiac cells. Our results, taken together, emphasize the
importance of syntrophins as multidomain scaffolds that link NaChs to
the DAPC and, hence, to the actin cytoskeleton and the extracellular
matrix.
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FOOTNOTES |
Received June 19, 1997; revised Oct. 13, 1997; accepted Oct. 20, 1997.
This work was supported by grants from the National Institutes of
Health (S.R.L., J.H.C., R.S., and S.C.F.) and the Muscular Dystrophy
Association (R.S. and S.C.F.). S.H.G. is supported by a Human Frontier
Science Program Organization Postdoctoral Fellowship. We thank Dr.
Brian Kay and Stacy Sekely (University of North Carolina at Chapel
Hill) for providing APC and Fas peptides, Karen Christopherson, Jay
Brenman, and Dr. David Bredt (University of California at San
Francisco) for nNOS PDZ domain fusion proteins, and Dr. James Trimmer
(State University of New York at Stony Brook) for antibodies to
K+ channel subunits. We especially thank Dr.
Christian R. Lombardo of the University of North Carolina
Macromolecular Interactions Facility for help with the collection and
analysis of surface plasmon resonance data and Donna Krzemien for
technical assistance. Finally, we thank the reviewers for their
comments and suggestions.
Correspondence should be addressed to Dr. Stanley C. Froehner or Dr.
Robert Sealock, Department of Physiology, University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599-7545
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Top
Abstract
Introduction
