From the
The voltage-dependent chloride channel ClC-1 stabilizes resting
membrane potential in skeletal muscle. Mutations in the ClC-1 gene are
responsible for both human autosomal recessive generalized myotonia and
autosomal dominant myotonia congenita. To understand the tissue
distribution and subcellular localization of ClC-1 and to evaluate its
role in an animal model of myotonia, antibodies were raised against the
carboxyl terminus of this protein. Expression of the 130-kDa ClC-1
protein is unique to skeletal muscle, consistent with its mRNA tissue
distribution. Immunolocalization shows prominent ClC-1 antigen in the
sarcolemma of both type I and II muscle fibers. Sarcolemma localization
is confirmed by Western analysis of skeletal muscle subcellular
fractions. The ADR myotonic mouse (phenotype ADR, genotype
adr/adr), in which defective ClC-1 mRNA has been identified,
is shown here to be absent in ClC-1 protein expression, whereas other
skeletal muscle sarcolemma protein expression appears normal.
Immunohistochemistry of skeletal muscle from ADR and other mouse models
of human muscle disease demonstrate that the absence of ClC-1 chloride
channel is a defect specific to ADR mice.
The voltage-dependent Cl
The first member of the ClC family of voltage-gated chloride
channels, ClC-0, was identified by expression cloning of Torpedo
electric organ cDNAs
(10) . Rat skeletal muscle ClC-1, which is
55% identical to ClC-0, is a 994-amino acid protein with a predicted
molecular mass of 110 kDa
(11) . Northern blot analysis of ClC-1
detected prominent mRNA expression in skeletal muscle, with weak
expression in kidney, liver, heart, and smooth muscle
(11) . The
apparent muscle-specific expression of the ClC-1 mRNA lies in stark
contrast to the ubiquitous epithelial and non-epithelial expression of
ClC-2
(12) . ClC-1 and ClC-2 sequences are 55% homologous, with
greatest divergence in the amino and carboxyl termini and between
domains D12 and D13. Both proteins are proposed to have 12
transmembrane domains, although a 13th potential transmembrane domain
is presumed to be entirely cytoplasmic, based on the inability of
carboxyl-terminal deletions to affect channel inactivation
(13) .
The ADR (``arrested development of righting
response'') mouse has frequently been used as a model of human
recessive autosomal myotonia. Human myotonias are similar to this mouse
model on the basis of hyperexcitability of the plasma membrane and a
physiological defect in sarcolemmal Cl
Although the voltage-dependent chloride
channel ClC-1 has been well studied electrophysiologically and
genetically due to the involvement of patients with defects in this
gene, there has been very little biochemical characterization of this
protein. In this study, the 130-kDa voltage-dependent chloride channel
ClC-1 is shown to be localized to the sarcolemma by immunofluorescence
and subcellular fractionation of skeletal muscle membranes. Protein
expression appears to be limited to skeletal muscle. By
immunocytochemistry, we demonstrate the specific absence of the
chloride channel ClC-1 protein in ADR mice.
A single immunoreactive protein of approximately 130 kDa is
recognized in skeletal muscle microsomes with antibodies prepared
against the deduced 15 carboxyl-terminal amino acids of ClC-1 chloride
channel (Fig. 1). This antibody also recognizes the
BSA-conjugated peptide, but not unconjugated BSA. The skeletal muscle
tissue specificity of ClC-1 is demonstrated by the inability of the
ClC-1 carboxyl-terminal antibody to recognize protein in membranes of
any other tissue (data not shown). The absence of ClC-1 protein in
tissues other than skeletal muscle is in accordance with its mRNA
expression
(11) , although small amounts of mRNA were detected
in liver, kidney, and heart, but were not detected by Western blot
analysis of crude membrane preparations. The carboxyl-terminal 15 amino
acids of rat skeletal muscle are unique to ClC-1 and are not found in
any other chloride channels identified to date, including the Torpedo
electroplax ClC-0
(10) , the ubiquitously expressed ClC-2
(12) , or the K-1 of the inner medulla of the kidney
(22) .
Establishing
the distribution of chloride-conducting channels in the sarcolemma and
transverse tubule membrane system is useful in understanding the action
potential repolarization phase and action potential propagation,
especially as it relates to disease states such as myotonia
(23, 24, 25) . Labeling of transverse skeletal
muscle cryosections with carboxyl-terminal ClC-1 antibody reveals
exclusive staining of the cell periphery, suggestive of a sarcolemmal
localization (Fig. 2). Staining was uniformly distributed along
the sarcolemmal surface of all fiber types, supporting the presence of
ClC-1 in both type I and II muscle fibers. Specific staining was able
to be competed by preincubation of the antibody with the 15-amino acid
peptide against which this antibody was raised.
Recent evidence has suggested that a highly conserved consensus site
for N-linked glycosylation between domains D8 and D9 is
glycosylated in vitro (31) . Biochemical
deglycosylation with glycanase F resulted in a small, but difficult to
detect and reproduce, mobility shift of 2-3 kDa, which may
represent deglycosylation of a single N-glycosylation site
(data not shown). Under identical conditions, complete deglycosylation
was observed in the heavily glycosylated voltage-dependent
Ca
This is the first biochemical
characterization of the skeletal muscle voltage-dependent chloride
channel ClC-1. Although an earlier study identified a 110-120-kDa
protein in purified sarcolemma fractions of rabbit skeletal muscle that
correlated with chloride channel activity and indanyloxyacetic acid
binding, this protein could not be unambiguously identified as ClC-1
without the use of specific antibodies
(32) . Interestingly, a
60-kDa protein also copurified in the fractions with chloride channel
activity, suggesting the presence of an auxiliary subunit such as has
been found to associate with most other voltage-gated channels. Further
purification of the chloride channel complex should clarify its
multimeric structure.
While several investigators have identified
and examined the electrophysiological properties of mutant ClC-1
proteins expressed in Xenopus oocytes, a biochemical approach
will allow us to determine whether ClC-1 may be regulated by the
serine/threonine protein kinase implicated in the pathogenesis of
myotonic dystrophy. Also, since we have identified the absence of ClC-1
protein in ADR mice, it is probable that some forms of human autosomal
recessive myotonia congenita may also be associated with a deficiency
of this protein. Determination of ClC-1 expression by
immunofluorescence microscopy of muscle biopsy may aid genetic analysis
in the diagnosis of patients with myotonia congenita.
We thank M. J. Mullinnix for his expert technical
assistance and S. L. Roberds, D. R. Witcher, and R. H. Crosbie for
comments on this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channel
ClC-1
(
)
is responsible for maintaining resting
membrane potential in skeletal muscle
(1) . Hyperexcitability of
muscle membranes following voluntary contractions is a hallmark of
myotonia, suggesting either an increase in sodium conductance and/or a
decrease in chloride conductance. Blockage of Cl
conductance elicits myotonia in experimental animals
(2, 3) . Moreover, mutations in the ClC-1 gene have been
shown to be responsible for hereditary myotonia in mouse
(4, 5) and human
(6, 7, 8, 9) .
conductance
(14) . The insertion of a transposon of the Etn family
after the D9 coding sequence of the ClC-1 gene leads to aberrant small
mRNA fragments which appear insufficient to encode a functional
Cl
channel
(4) . However, no study has
examined ClC-1 protein expression in the ADR mouse or in any human with
this genetic disease.
Antibody Preparation
A peptide representing the
15 carboxyl-terminal amino acids (amino acids 980-994) of the
cloned rat skeletal muscle chloride channel ClC-1 was prepared by the
HHMI Peptide Facility (Washington University, St. Louis, MO) as an
NH-terminal p-benzoylbenzoic acid photoprobe.
Peptide was conjugated to keyhole limpet hemocyanin and injected into
rabbits as described
(15) . Peptide was also conjugated to
bovine serum albumin (BSA) for immunoblot analysis. Antibodies to the
subunit of the voltage-dependent Ca
channel were prepared against an 18-amino acid peptide
corresponding to residues 839-856 of rabbit skeletal muscle
according to De Jongh et al. (16) . Peptide was coupled
to keyhole limpet hemocyanin and BSA using the bifunctional coupling
agent m-maleimidobenzoic acid- N-hydroxysuccinimide
ester through an NH
-terminal cysteine residue on the
peptide. Rabbit polyclonal antibodies were affinity-purified against
immobilized strips of COOH-terminal peptide coupled to BSA as described
previously
(17) . Na,K-ATPase monoclonal antibody was provided
by Dr. Kathleen Sweadner (Harvard University).
Membrane Preparation
KCl-washed membranes were
prepared from three age-matched control (?/+) and adr/adr mice as described previously
(18) . Sarcolemma membranes
were separated from KCl-washed microsomes by the wheat germ
agglutination method described by Ohlendieck and Campbell
(18) .
SDS-PAGE and Immunoblotting
SDS-PAGE
(19) was carried out on 3-12% gradient gels in the
presence of 1% 2-mercaptoethanol (unless otherwise indicated) and
stained with Coomassie Blue or transferred to nitrocellulose
(20) . Molecular weight standards were purchased from Life
Technologies, Inc. Nitrocellulose transfers were stained with
polyclonal antibodies, affinity-purified antibodies, or monoclonal
antibodies as described previously
(21) . Affinity-purified
antibodies were used at a 1:50 dilution for immunoblotting.
Immunofluorescence
Cryosections (7 µm) of
quadriceps muscle age-matched mice were incubated with nondiluted
affinity purified COOH-terminal ClC-1 antibody or 1:1,000 dilution of
mAb XIXC2 (specific for dystrophin). Sections had been initially
blocked for 1 h in 5% BSA in phosphate-buffered saline. Incubation in
1:200 biotinylated-labeled goat anti-rabbit IgG secondary for 1 h was
followed by incubation for 30 min with 1:1,000 diluted
fluorescein-conjugated streptavidin (Jackson ImmunoResearch
Laboratories). Conventional fluorescence microscopy was carried out
with an Axioplan photomicroscope. Samples were prepared uniformly, and
photographs were taken under identical conditions with same exposure
times.
Figure 1:
Characterization of rabbit antibody to
the carboxyl terminus of a rat skeletal muscle chloride channel ClC-1.
A, the 15 carboxyl-terminal amino acids of the cloned rat
skeletal muscle chloride channel (10). B, shown are a
Coomassie Blue-stained gel ( left panel) and a nitrocellulose
transfer ( right panel) stained with affinity-purified rabbit
anti-ClCh antibodies. Lane 1, BSA (3 µg); lane 2,
BSA-conjugated to carboxyl-terminal peptide (3 µg); lane
3, rabbit KCl-washed skeletal muscle microsomes (150 µg).
Arrows indicate the positions of the chloride channel
( ClCh) and the BSA-conjugated COOH-terminal peptide
( BSA-peptide). Molecular weight markers are indicated on the
left.
Cross-reactivity of the carboxyl-terminal antibody
with rabbit, dog, mouse, hamster, guinea pig, and human skeletal muscle
ClC-1 suggests conservation of at least a portion of this
carboxyl-terminal sequence in mammals (data not shown). Although this
antibody did not appear to react with nonmammalian tissue, an
exhaustive test of various species was not performed.
Figure 2:
Sarcolemma distribution of ClC-1 in mouse
skeletal muscle. Immunofluorescence microscopy of mouse skeletal muscle
transverse cryosections stained with affinity-purified chloride channel
carboxyl-terminal antibody in the absence and presence of competing
carboxyl-terminal peptide. Antibody specifically labeled the cell
periphery, whereas specific staining was not observed in the internal
regions of muscle fibers.
Confirmation of
ClC-1 distribution by fractionation of skeletal muscle membranes into
purified sarcolemma and WGA-void fraction, which contains both
transverse tubules and sarcoplasmic reticulum, demonstrates the
presence of ClC-1 in the sarcolemma and its absence in the WGA-void
fraction (Fig. 3). This is in contrast to the voltage-dependent
Cachannel
subunit, which is
characteristically localized to the transverse tubules and is thus
enriched in the WGA-void
(18, 26) . These results differ
dramatically from previous physiological localization of skeletal
muscle chloride conducting channels. Chloride conductance in mammalian
muscle was previously shown to be similar on surface and tubular
membranes
(24, 27) , which when corrected for surface
area suggested that up to 80% of chloride conductance may be associated
with transverse tubules. Although ClC-1 does not appear to be present
in transverse tubules by both immunofluorescence and cell
fractionation, it is likely that either a distinctly different chloride
conducting channel or a ClC-1 splice variant which differs at the
carboxyl terminus may be present in transverse tubules. The defects in
human and animal models of myotonia identified to date, however, appear
to be specific for the sarcolemma protein ClC-1. The sarcolemma
localization of ClC-1 has striking implications for understanding the
mechanisms of myotonic properties such as depolarizing afterpotentials
which allow repetitive firing of action potentials and tubular
depolarization.
Figure 3:
Immunoblot analysis of purified sarcolemma
preparation. Coomassie Blue-stained gel ( left panel) and
nitrocellulose transfer ( middle and right panel)
stained with affinity-purified rabbit chloride channel ( ClCh)
antibodies and affinity-purified voltage-dependent Cachannel
(
) subunit antibodies
and detected by ECL (Amersham Corp.). First lane, 150 µg
of mouse crude surface membranes; second lane, 150 µg of
sarcolemma; third lane, 150 µg of WGA-void fraction.
Molecular weight standards are indicated on the left. Low
molecular weight fragments identified by affinity-purified chloride
channel antibodies are degradation products.
The single channel properties of ClC-0 and the
dominant negative effect of Thomsen-type myotonia congenita ClC-1
mutations predict a homodimeric or homotetrameric structure for this
chloride channel gene family
(8, 28) . To investigate
whether this oligomeric structure may be due to disulfide linked
monomers, crude surface membranes were run under reduced and nonreduced
conditions. Although the characteristic 25-kDa mobility shift was seen
in the voltage-dependent Cachannel
subunit on dissociation of the disulfide linked
subunit
(29) , disulfide reduction appeared to have no effect on ClC-1
mobility (data not shown). The absence of a mobility shift on disulfide
bond reduction suggests that, if this channel is indeed a
homooligomeric structure, it is not comprised of disulfide-linked ClC-1
monomers. However, voltage-dependent K
channels form a
well established homotetramer without intersubunit disulfide bonds, and
the chloride channel oligomeric structure may be similar
(30) .
channel
subunit. However, the
majority of 1% digitonin solubilized ClC-1 is not retained by WGA
affinity chromatography (data not shown) and no differences are seen on
concanavalin A peroxidase staining of control and ADR mouse skeletal
muscle (Fig. 4 A). Experiments on purified chloride
channel complex may further elucidate the presence and location of
glycosylated residues. Glycosylation also does not account for the
difference in the 110 kDa predicted molecular mass based on sequence
analysis
(11) and the 130 kDa calculated for the native
protein. Anomalous SDS-PAGE mobility of ClC-1 may be due to the
multiple membrane-spanning regions or the highly negatively charged
domain between D12 and D13.
Figure 4:
Specific deficiency of ClC-1 chloride
channel protein in ADR mice. A, immunoblot analysis of control
and adr/adr mouse muscle membranes. Shown are a Coomassie
Blue-stained gel ( CB) and identical immunoblots stained with
affinity-purified anti-chloride channel antibodies ( ClCh),
affinity-purified rabbit antibody to the COOH terminus of dystrophin
( DYS), mAb IIF7 to the subunit of the
skeletal muscle dihydropyridine receptor ( DHPR), mAb McB2 to
the Na
/K
-ATPase, and concanavalin
A-peroxidase ( ConA). Lanes 1 represent (?/+)
control membranes and lanes 2 represent adr/adr
membranes. Protein amounts (from left to right) are
100, 200, 100, 100, 50, and 100 µg. B, immunofluorescence
labeling of mouse skeletal muscle with chloride channel (ClC-1) and
dystrophin antibodies. Transverse cryosections were labeled for
indirect immunofluorescence with affinity-purified anti-chloride
channel antibodies ( ClCh) and mAb XIXC2 against dystrophin
( DYS). Cryosections are from skeletal muscle of age-matched
control (?/+), adr/adr, dfw/dfw, dy/dy, and mdx
mice.
Although ClC-1 protein expression is
predicted to be absent in ADR mice based on phenotypic and genetic
analysis
(11) , here we confirm its absence in skeletal muscle
membranes. ClC-1 protein is specifically absent in adr/adr
mouse skeletal muscle as compared with age-matched controls
(Fig. 4 A). The defect is specific for ClC-1, as
expression of dystrophin, dihydropyridine receptor, and
Na/K
-ATPase all appear to be normal
in ADR mice. Immunofluorescence microscopy also demonstrates the
specific absence of ClC-1 sarcolemma staining in ADR mice
(Fig. 4 B). Dystrophin, which is also localized to the
sarcolemma, is expressed normally in the myofibers of ADR mice. Loss of
ClC-1 immunoreactivity does not appear to be a nonspecific marker of
muscle disease, as this protein is expressed at normal levels in dy/dy,
dfw/dfw, and mdx mice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.