Localization of sodium channel subtypes in adult rat skeletal muscle using channel-specific monoclonal antibodies

Five monoclonal antibodies specific for the 260 kDa subunit of the rat skeletal muscle sodium channel were used to probe the distribution of this channel in adult muscle. All the antibodies reacted with the surface membrane of fast- and slow-twitch fibers in the rat anterior tibial and soleus muscles. Immunoreactivity was also present in the endplate region; this was significantly more intense than that in the surrounding extrajunctional membrane. At the electron microscopic level, this junctional immunoreactivity could be traced uniformly throughout the secondary folds of the post-synaptic membrane. Three of the monoclonal antibodies (A/B2, F/E4, and I/E3) exhibited an additional distinct immunoreactivity pattern, staining the interior of selected fibers in the anterior tibial muscle that were subsequently identified as slow-twitch fibers. An identical reactivity pattern was observed with most of the soleus muscle fibers. In longitudinal sections of slow fibers examined at the light microscopic level, transversely oriented, regularly spaced doublets of fluorescence were localized at the junction of the A and I bands in each sarcomere. In permeabilized slow fibers exposed to A/B2 and examined at the electron microscopic level, internal reactivity was associated exclusively with the membranes of the T-tubular system. A/B2 also strongly stained a transversely oriented pattern within cardiac muscle fibers exhibiting the characteristics of the T-tubular system in that tissue. We conclude that at least 3 subpopulations of sodium channels are present in adult skeletal muscle: those in the sarcolemma of fast and slow fibers, those in slow-twitch fiber T-tubular membranes, and those in the T-tubular system of fast fibers. The channels in the slow fiber T-system apparently share common epitopes with those in the T-system of cardiac fibers.     

Expression of sodium channel subtypes during development in rat skeletal muscle.

This study contrasts the developmental patterns of expression of 2 subtypes of the voltage-dependent sodium channel in rat muscle that are differentiated by their immunoreactivity with monoclonal antibodies raised to the purified muscle sodium channel protein. One subtype is found in the transverse tubular (T) system of slow twitch fibers as well as the plasma membrane of fast and slow twitch fibers in the anterior tibial and soleus muscles. The second is present in the plasma membrane in all fibers of both muscles. The transverse tubular subtype exhibits 2 immunocytochemical staining patterns within muscle fibers, reticular and homogeneous, which may represent labeling of the developing T tubular system and of a cytoplasmic pool of alpha subunits of the sodium channel respectively. The reticular pattern eventually disappears in fast twitch fibers but persists into the adult stage in slow twitch fibers. The homogeneous pattern is also seen with antibodies to the plasma membrane subtype and disappears in early development as immunoreactivity to both subtypes gradually appears in the surface membrane. A reticular pattern is never seen with the plasma membrane subtype. The factors that modulate the expression of these subtypes is unknown.     

Hydrocortisone influences voltage-dependent L-type calcium channels in cultured human skeletal muscle

The glucocorticoid hydrocortisone (HC), applied for up to 2 weeks to either aneurally or innervated cultured human muscle, produced 2-fold increase of the number of dihydropyridine ([³H]PN200-110) binding sites. The K⁺ -induced, nifedipine-inhibited Ca²⁺ uptake was increased 40%. The effect of HC was concentration- and time-dependent. [³H]PN200-110 affinity for its receptor was not affected by HC treatment. HC did not exert significant influence on the total amount of protein, CK activity, and the number of myotubes. These results indicate that voltage-dependent L-type Ca²⁺ channel expression in human muscle is regulated by glucocorticoid. © 1995 Wiley-Liss, Inc.     

Acetazolamide acts directly on the human skeletal muscle chloride channel

Acetazolamide, a carbonic anhydrase inhibitor, is used empirically in neuromuscular diseases with episodic ataxia, weakness, and myotonia, although not all of the mechanisms responsible for its therapeutic effects are understood. To elucidate whether acetazolamide acts directly on the human skeletal muscle voltage-gated chloride channel (ClC-1), which is associated with myotonia, we evaluated the effects of acetazolamide on ClC-1 expressed in cultured mammalian cells, using whole-cell recording. Acetazolamide significantly shifted the voltage dependency of the open probability (P(o)) toward negative potentials in a dose-dependent manner, resulting in an increase of chloride conductance at voltages near the resting membrane potential. This effect was attenuated when using a pipette solution containing 30 mmol/L Hepes. These results suggest that acetazolamide can influence the voltage-dependent opening gate of ClC-1 through a mechanism related to intracellular acidification by inhibiting carbonic anhydrase, and that the therapeutic effects of acetazolamide in neuromuscular diseases may be mediated by activation of ClC-1.     

Paramyotonia congenita: The R1448P Na+ channel mutation in adult human skeletal muscle

Twitch force and Na+ currents were investigated in a muscle biopsy specimen from a patient with paramyotonia congenita carrying the dominant Arg-1448-Pro mutation in the skeletal muscle sodium channel. Cooling of the muscle fibers caused sustained membrane depolarization that resulted in reduced twitch force. Membrane repolarization, produced by a K+ channel opener, partly prevented and antagonized the drop in twitch force. Patch-clamp recordings on sarcolemmal blebs revealed a distinctly slower Na⁺ current decay on paramyotonia congenita muscle compared to control muscle. In addition, patches with mutant Na⁺ channels showed a significantly higher frequency of steady-state openings, which increased with cooling. Activation of mutant channels was not affected, whereas the steady-state inactivation curve was shifted by ?5 mV and showed less voltage dependence. We suggest that the weakness of cooled muscle can be explained by a combination of the increased steady-state Na⁺ current and the left-shifted inactivation curve.     

Primary skeletal muscle involvement in chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is a hereditary disease characterized by involuntary movements and amyotrophy with elevation of serum creatine kinase. Although skeletal muscle involvement in ChAc has been suggested, the mechanism remains unclear. To investigate chorein abnormalities of the skeletal muscles of ChAc patients with an apparently heterozygous VPS13A mutation compared with those of other hereditary choreic diseases, we performed histological and immunohistochemical studies of the skeletal muscles from 3 ChAc, 1 Huntington's disease (HD), 1 McLeod syndrome (MLS), and 1 normal control (NC) with 2 originally generated anti-chorein antibodies. Chorein immunoreactivities in HD, MLS, and NC were found linearly along the sarcolemma and appeared as speckles in the sarcoplasma, but those in ChAc were uneven and discontinuous along the sarcolemmas and increased in the sarcoplasma especially in type I fibers. This histological observation suggests chorein abnormalities of skeletal muscles might be associated with primary involvement of skeletal muscles in this disorder.     

Paramyotonia congenita and hyperkalemic periodic paralysis are linked to the adult muscle sodium channel gene.

The hyperkalemic periodic paralyses are a clinically heterogeneous group of autosomal dominant syndromes characterized by episodic paralysis associated with an elevated serum potassium level. Affected individuals in the same family tend to have homogeneous symptom complexes, although phenotypic variation is present among different families. For example, myotonia is absent in some pedigrees, present in others, and, in a third variant, paramyotonia congenita, myotonia coexists with cold-induced paralysis. Electrophysiological studies have demonstrated variant-specific abnormalities in skeletal muscle membrane sodium conductance. We tested the hypothesis that hyperkalemic periodic paralysis (without myotonia) and paramyotonia congenita are tightly linked to the tetrodotoxin-sensitive adult skeletal muscle sodium channel gene on chromosome 17q23-25 in two large pedigrees. The DNA polymorphisms detected in the growth hormone skeletal muscle sodium channel complex (GH1-SCN4A) and by flanking polymorphic markers (D17S74 and D17S40) demonstrated no recombinants between the disease phenotypes and this complex. Phenotypic variation in the hereditary hyperkalemic periodic paralyses may result from allelic heterogeneity at the tetrodotoxin-sensitive adult skeletal muscle sodium channel locus.     

Human skeletal muscle sodium channelopathies

Abstract Ion channels are transmembrane proteins that allow ions to flow in or out of the cell. Sodium and potassium channel activation and inactivation are the basis of action potential’s production and conduction. During the past 15 years, ion channels have been implicated in diseases that have come to be known as the channelopathies. Over 30 mutations of the muscle channel gene SCN4A, which encodes the muscle voltage-gated sodium channel, have been described and associated with neuromuscular disorders like hypo- and hyper-kalaemic periodic paralyses (hypoPP and hyperPP), paramyotonia congenita, sodium channel myotonias and congenital myasthenic syndrome. Different mutations within the same gene (SCN4A) cause distinct clinical disorders, while mutations in different channel genes may result in similar phenotypes. In addition, identical sodium channel mutations can result in different clinical phenotypes (hyperPP or paramyotonia) in different members of the same family, suggesting that the genetic background and perhaps other epigenetic factors may influence the clinical expression of a particular mutation. This article reviews the clinical features of the skeletal muscle sodium channel diseases and highlights the phenotypic or genetic overlap in these disorders.     

A novel mutation in the gene for the adult skeletal muscle sodium channel alpha-subunit (SCN4A) that causes paramyotonia congenita of von Eulenburg.

BACKGROUND: Paramyotonia congenita (PMC) of von Eulenburg is an autosomal dominant muscular disease characterized by exercise- and cold-induced myotonia and weakness. To date, 18 missense mutations in the adult skeletal muscle sodium channel alpha-subunit (SCN4A) gene have been identified to cause a spectrum of muscular diseases, including PMC of von Eulenburg, PMC without cold paralysis, potassium-aggravating myotonia, and hyperkalemic periodic paralysis. However, no obvious correlations can be made between the location or nature of amino acid substitutions in SCN4A and its clinical phenotypes. OBJECTIVE: To describe clinical and genetic features of a family with PMC of von Eulenburg. RESULTS: A Japanese family with cold-induced myotonia and weakness was diagnosed as having PMC of von Eulenburg. This phenotype was identified to be caused by a novel mutation that substituted a glutamic acid residue for a highly conserved glycine residue in the fourth transmembrane segment (S4) of domain IV. This predicted a decrease in positive charge specific for the S4. CONCLUSION: In addition to the G1456E identified in this study, 4 mutations that cause a decrease in positive charge in the S4/D4 are associated with the phenotype of PMC of von Eulenburg. This provides an important genotype-phenotype correlation in sodium channelopathies.     

Different localization of dystrophin in developing and adult human skeletal muscle

Duchenne and Becker muscular dystrophy are caused by defects in dystrophin synthesis. Using affinity-purified polyclonal anti-dystrophin antibodies, we have studied immunohistochemically the subcellular localization of dystrophin in embryonic, fetal, and adult human skeletal muscle. In the embryonic stages dystrophin first appears in the sarcoplasm at the peripheral ends of the myotubes, immediately adjacent to the tendons, whereas in fetal stages dystrophin is found throughout the entire myofibers. In agreement with literature data, in adult muscle dystrophin expression was found to be restricted to the sarcolemma. The sarcoplasmic localization in embryonic and fetal tissue and the sarcolemmal localization of dystrophin in mature muscle suggests the accumulation of dystrophin in the cytoplasm prior to its integration into the membrane. These results increase our knowledge of the ontogenesis of dystrophin and may lead to a better understanding of the great diversity in pathological cases of Duchenne and Becker muscular dystrophy.     

Effect of mexiletine on sea anemone toxin-induced non-inactivating sodium channels of rat skeletal muscle: a model of sodium channel myotonia.

The sea anemone toxin ATX II impairs skeletal muscle sodium channel inactivation, mimicking the persistent inward current observed in patients suffering from sodium channel myotonia. Mexiletine has beneficial effects on myotonia. To verify the efficiency of the drug on persistent inward current, we investigated the effect of 50 microM R(-)-mexiletine on sodium channels in cell-attached patches of rat skeletal muscle fibres, in the absence or presence of 2 microM ATX II. With the toxin, a proportion of channels displayed remarkable abnormal activity lasting the entire depolarisation, which resulted in a persistent inward current that represented up to 2.0% of the peak current. Mexiletine reduced by 75% the peak current elicited by depolarisation from -100 to -20 mV. This was due to the reduction by 60% of the maximal available peak current Imax and to the negative shift by -7 mV of steady-state inactivation. Mexiletine also greatly decreased the late current, but the effect was limited to 60% of reduction, comparable to that on Imax. Therefore mexiletine was able to block the ATX II-modified sodium channels, inhibiting the myotonia-producing persistent inward current.     

Cylindrical spirals in human skeletal muscle.

Muscle biopsies from two patients revealed that numerous type 2 fibers contained large abnormal areas filled with cylindrical spirals. The cytochemical profile of these cylindrical spirals was sufficiently characteristic that they could be distinguished from tubular aggregates. Their electron microscopic appearance was unmistakable. Their origin and significance are uncertain. The diverse nature of the patients' conditions (cramps and malignancy, and an unusual form of spinocerebellar degeneration) indicate that these abnormal structures are not disease specific.     

A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.

With our new "explant-reexplanting" technique, abundant growth of mature human muscle in long-term tissue culture was achieved,and with the "sandwich" technique several histochemical reactions were obtained on serial cross sections of the cultured fibers. An advanced degree of maturation but lack of differentiation into reciprocally staining fiber-types was demonstrated. For electron-microscopic and electronmicroscopic-histochemical study, a method was developed in which the embedded fibers of greatest potential interest were identified by light microscopy and punched out by our specially-designed hollow drill. This selection procedure is critically important when the goal is to study in cultured diseased human muscle: (1) successive stages of development and (2) certain structural changes that often occur only in some fibers and only in certain regions of those fibers. The electronmicroscopic-histochemical appearance of developing cultured muscle fibers correlated well with the fresh-frozen light microscopic histochemical cross-sections and longitudinal whole preparations of similar fibers.     

Immunocytochemical localization of the mammalian voltage-dependent sodium channel using polyclonal antibodies against the purified protein

Antibodies were raised in rabbits against the purified voltage-dependent sodium channel from rat skeletal muscle sarcolemma. The resultant antiserum reacted with the purified channel in a solid-phase radioimmunoassay and precipitated the sodium channel from a crude mixture of solubilized membrane proteins. Crude membrane proteins separated according to size under nondenaturing conditions by chromatography on Sepharose CL-6B contained a single peak of immunoreactivity that coincided with the native channel. On immunoblots of sarcolemmal membrane proteins, the antiserum reacted predominantly with a diffuse high molecular weight band that was comparable in migratory characteristics to the large glycoprotein subunit of the purified channel. Using immunocytochemical techniques, binding of this polyclonal antiserum was localized to the surface membrane of rat skeletal muscle. This staining was specifically blocked by pre-incubation of the antiserum with the purified channel protein. The antiserum also stained the surface membrane of rat cardiac muscle and the nodes of Ranvier in rat peripheral nerve. Species cross-reactivity was seen with mouse, human, and guinea pig skeletal muscle while chicken, rabbit, and frog muscle was not stained. The antiserum also reacted with the surface membranes of fetal rat muscle in tissue culture. These results indicate that sodium channels in adult mammalian skeletal muscle, cardiac muscle, and peripheral nerve and in fetal muscle in culture all share common antigenic determinants. The antiserum should prove useful for topographical studies of sodium channel distribution in these tissues.     

Two subtypes of sodium channel with tetrodotoxin sensitivity and insensitivity detected in denervated mammalian skeletal muscle

The action potential (AP) in most nerve and muscle preparations depends upon nanomolar concentrations of the neurotoxins saxitoxin (STX) and tetrodotoxin (TTX). In some excitable tissues lacking mature innervation, a toxin-resistant AP has been described by electrophysiological results. However, multiple attempts to detect corresponding toxin-resistant Na channels with radiolabelled STX and TTX have been unsuccessful. We report here the detection of Na channels with low-affinity binding of STX and TTX, accounting for 50-60% of the Na channels in rat hindlimb muscle 4-5 days after denervation.     

Effective adenovirus-mediated gene expression in adult murine skeletal muscle.

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the beta-actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express beta-galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SR alpha viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5- to 6-week-old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.