Expression of sodium channel subtypes during development in rat skeletal muscle (original) (raw)
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Sodium Channel NaV1.5 Expression is Enhanced in Cultured Adult Rat Skeletal Muscle Fibers
The Journal of Membrane Biology, 2010
This study analyzes changes in the distribution, electrophysiological properties, and proteic composition of voltage-gated sodium channels (Na V) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na V 1.5 channel type was observed. This was confirmed by an increase in TTXresistant channels and by Western blot test. These channels exhibited increased activation time constant (s m) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na V 1.5 was increasing progressively after denervation. By realtime polymerase chain reaction, we found that the expression of b subunits was also modified, but only after 7 days of culture: increase in b 1 without b 4 modifications. b 1 subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, s h was back to normal and s m still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na V 1.5 channels and the involvement of b subunits in the regulation of sodium current and fiber excitability.
Developmental appearance of sodium channel subtypes in rat skeletal muscle cultures
Journal of neurochemistry, 1986
22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin 11. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one-or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive ( K = 0.6-5.0 X lo-' M ) and an insensitive population (Ki = 3.3-4.9 X In primary rat muscle cultures, the sensitivity of the toxinstimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X lo-' M ) . A single population was also found in 6-day-old cultures (Ki = 5.3 X
Functional expression and properties of the human skeletal muscle sodium channel
Pflügers Archiv, 1994
Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the a-subunit of the adult human skeletal muscle Na + channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injected Xenopus oocytes and from transiently transfected tsA201 cells. The Na + currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na + currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na + currents by the extracellular toxins tetrodotoxin (TTX) and ~t-conotoxin (~tCTX) was measured. The ICso values were 25 nM (TTX) and 1.2 gM (gCTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkMl, but the potency of ktCTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.
Biochemistry of Sodium Channels from Mammalian Muscle
Annals of the New York Academy of Sciences, 1986
Mammalian skeletal muscle, like most nerve and muscle cells, generates action potentials mainly through transient changes in the sodium conductance of its surface membranes. The voltage-sensitive sodium channel that controls these time-and potential-dependent currents has been isolated from the surface membranes of rat skeletal muscle and from the T-tubular membranes of rabbit skeletal muscle.'-3 In its physiological and biophysical properties, this sodium channel closely resembles that found in most nerve membranes, and recent work suggests that this resemblance holds for many of the molecular properties of the isolated channel protein as weL4 MOLECULAR PROPERTIES OF THE ISOLATED MUSCLE CHANNEL When solubilized in nonionic detergents such as NP-40 or Lubrol PX, the sodium 179
Modulation of the skeletal muscle sodium channel α-subunit by the β1 -subunit
FEBS Letters, 1993
Co-expression of cloned sodium channel p,-subunit with the rat skeletal muscle-subunit (Q) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from cc&,, indicating that B, restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel /$-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' B, cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na' channel a-subunits in brain, skeletal muscle and heart may share a common /J-subunit.
Neuron, 1993
Transcripts homologous to the rat brain sodium channel p subunit CR,) are prominently expressed in both innervated and denervated adult skeletal muscle and in heart, but not in neonatal skeletal or cardiac muscle. Regulation of f3, mRNA expression closely parallels that of SkMl a during development, after denervation in adult muscle, and in primary muscle culture, but does not follow SkM2 expression under any condition examined. In oocytes, g+ interacts functionally with SkMl to modulate the abnormally slow inactivation kinetics observed with this a subunit expressed alone. We conclude that a common PI subunit is expressed in skeletal muscle, heart, and brain and that in skeletal muscle, this subunit is specifically associated with the SkMl, rather than the SkM2, sodium channel isoform.
Biochemical and Biophysical Research Communications, 1992
The ammo acid sequence of the sodium channel (Y subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the (r subunit from rat adult skeletal muscle and 70% identity to the (Y subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene. 0 1992 Academic Press, Inc.
Modulation of the skeletal muscle sodium channel alpha-subunit by the beta 1-subunit
FEBS Letters
Co-expression of cloned sodium channel beta 1-subunit with the rat skeletal muscle-subunit (alpha microI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from alpha microI/beta 1, indicating that beta 1 restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel beta 1-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' beta 1 cDNA to skeletal muscle and heart mRNA indicated that the different Na+ channel alpha-subunits in brain, skeletal muscle and heart may share a common beta 1-subunit.