Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA

There are dihydropyridine (DHP)-sensitive calcium currents in both skeletal and cardiac muscle cells, although the properties of these currents are very different in the two cell types (for simplicity, we refer to currents in both tissues as L-type). The mechanisms of depolarization-contraction coupling also differ. As the predominant voltage-dependent calcium current of cardiac cells, the L-type current represents a major pathway for entry of extracellular calcium. This entry triggers the subsequent large release of calcium from the sarcoplasmic reticulum (SR). In contrast, depolarization of skeletal muscle releases calcium from the SR without the requirement for entry of extracellular calcium through L-type calcium channels. To investigate the molecular basis for these differences in calcium currents and in excitation-contraction (E-C) coupling, we expressed complementary DNAs for the DHP receptors from skeletal and cardiac muscle in dysgenic skeletal muscle. We compared the properties of the L-type channels produced and showed that expression of a cardiac calcium channel in skeletal muscle cells results in E-C coupling resembling that of cardiac muscle.     

Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle

The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.     

Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs

The skeletal muscle dihydropyridine (DHP) receptor is essential in excitation-contraction (EC) coupling. The receptor is postulated to be the voltage sensor giving rise to the intramembrane current, termed charge movement. We have now tested this hypothesis using myotubes from mice with the muscular dysgenesis mutation, which alters the skeletal muscle DHP receptor gene and prevents its expression. Our results indicate that charge movement is deficient in dysgenic myotubes but is fully restored following injection of an expression plasmid carrying the rabbit skeletal muscle DHP receptor complementary DNA, strongly supporting the hypothesis that the DHP receptor is the voltage sensor for EC coupling in skeletal muscle. Additionally, our data obtained for normal and chimaeric DHP receptor constructs demonstrate that DHP receptors with widely differing abilities to function as calcium channels and to mediate EC coupling produce very similar charge movements.     

Induction of calcium currents by the expression of the alpha 1-subunit of the dihydropyridine receptor from skeletal muscle

The dihydropyridine (DHP) receptor purified from skeletal muscle comprises five protein subunits (alpha 1, alpha 2, beta, gamma and delta) and produces Ca2+ currents that are blocked by DHPs. Cloning of the alpha 1- and alpha 2-subunits, the former affinity-labelled by DHP, has shown that the alpha 1-subunit is expressed in skeletal muscle alone, whereas the alpha 2- and delta- subunits are also expressed in other tissues. Although the transient expression of the alpha 1-subunit in myoblasts from dysgenic mice (but not in oocytes) has been demonstrated, the use of these expression systems to determine the function of the alpha 1- subunit is complicated by the presence of endogenous Ca2+ currents, which may reflect the constitutive expression of proteins similar to the alpha 2-, beta-, gamma- and/or delta-subunits. We therefore selected a cell line which has no Ca2+ currents or alpha 2- subunit, and probably no delta-subunit for stable transformation with complementary DNA of the alpha 1- subunit. The transformed cells express DHP-sensitive, voltage-gated Ca2+ channels, indicating that the minimum structure of these channels is at most an alpha 1 beta gamma complex and possibly an alpha 1- subunit alone.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.     

Slow inward calcium currents have no obvious role in muscle excitation-contraction coupling

It has been proposed that an influx of calcium ions into twitch muscle fibres during an action potential might initiate contraction. However, when external Ca2+ is lowered to 10(-8) M with EGTA, the fibres can produce normal twitches for many minutes. Nevertheless, a clear Ca2+ influx during contraction has been demonstrated, and it has been found that phasic skeletal muscle has an inward calcium current (ICa) which can give rise to calcium spikes. In certain conditions, a reduction in external Ca2+ with 80-90 mM EGTA results in reversible blockade of excitation-contraction (e-c) coupling, leading some authors to suggest that extracellular Ca2+ moved into the myoplasm due to ICa may be involved in the e-c coupling mechanism that triggers contraction. This proposition was further supported by the localization of ICa in the T-system, which circumvented the problem of the delay due to calcium diffusion from the surface membrane. We have now investigated whether ICa has a clear role in initiating or sustaining contractions in twitch muscle fibres. Our approach was to decrease or eliminate ICa with the calcium-blocking agent diltiazem (Herbesser) and to see how the twitch, tetanic and potassium-contracture tensions were affected. We found that ICa could be decreased or cancelled with the calcium-blocking agent, but that the same concentration of the drug potentiated the twitch, tetanus and contractures. We conclude, therefore, that ICa has no role in e-c coupling. A preliminary report of these results has been presented elsewhere.     

Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics.

Membrane depolarization causes many kinds of ion channels to open, a process termed activation. For both Na+ channels and Ca2+ channels, kinetic analysis of current has suggested that during activation the channel undergoes several conformational changes before reaching the open state. Structurally, these channels share a common motif: the central element is a large polypeptide with four repeating units of homology (repeats I-IV), each containing a voltage-sensing region, the S4 segment. This suggests that the distinct conformational transitions inferred from kinetic analysis may be equated with conformational changes of the individual structural repeats. To investigate the molecular basis of channel activation, we constructed complementary DNAs encoding chimaeric Ca2+ channels in which one or more of the four repeats of the skeletal muscle dihydropyridine receptor are replaced by the corresponding repeats derived from the cardiac dihydropyridine receptor. We report here that repeat I determines whether the chimaeric Ca2+ channel shows slow (skeletal muscle-like) or rapid (cardiac-like) activation.     

Primary structure of the receptor for calcium channel blockers from skeletal muscle

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.     

Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor

The sequence of 5,037 amino acids composing the ryanodine receptor from rabbit skeletal muscle sarcoplasmic reticulum has been deduced by cloning and sequencing the complementary DNA. The predicted structure suggests that the calcium release channel activity resides in the C-terminal region of the receptor molecule, whereas the remaining portion constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum and the transverse tubule.     

Insulin receptor tyrosine kinase is defective in skeletal muscle of insulin-resistant obese mice

Obese syndromes of genetic origin or experimentally induced are characterized by resistance to insulin both in vivo (association of hyperglycaemia and hyperinsulinaemia) and in vitro. Thus, skeletal muscle of obese mice, which is the most important target organ for the action of insulin, displays a reduced response to insulin. This hormonal resistance cannot be explained by the moderate decrease in the number of insulin receptors found in obese animals. In fact, it is generally believed that a biochemical event occurring very early after binding of insulin to its receptor, which is the first step in insulin action, is defective in obesity. One of the earliest post-binding events so far recognized, and which is thought to have a key role in cellular signalling by the insulin receptor, is the insulin-stimulated phosphorylation of its receptor. In an effort to localize the defect responsible for the insulin resistance in obesity, we have studied the insulin receptor protein kinase activity and we show here that insulin receptors from skeletal muscles of insulin-resistant obese mice have an altered kinase activity for phosphorylation of both the receptor itself and of exogeneous substrates.     

运动与骨骼肌中的基因表达

通过献资料法阐述了运动对骨骼肌基因表达的影响,以便深入理解骨骼肌的工作原理,为客观指导运动训练提供依据．     

Nucleotide sequence of the rat skeletal muscle actin gene

The actins constitute a family of highly conserved proteins found in all eukaryotic cells. Their conservation through a very wide range of taxonomic groups and the existence of tissue-specific isoforms make the actin genes very interesting for the study of the evolution of genes and their controlling elements. On the basis of amino acid sequence data, at least six different mammalian actins have been identified (skeletal muscle, cardiac muscle, two smooth muscle actins and the cytoplasmic beta- and gamma-actins). Rat spleen DNA digested by the EcoRI restriction enzyme contains at least 12 different fragments with actin-like sequences but only one which hybridized, in very stringent conditions, with the skeletal muscle cloned cDNA probe. Here we describe the sequence of the actin gene in that fragment. The nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. There are five small introns in the coding region and a large intron in the 5'-untranslated region. Comparison of the structure of the rat skeletal muscle actin gene with available data on actin genes from other organisms shows that while the sequenced actin genes from Drosophila and yeast have introns at different locations, introns located at codons specifying amino acids 41, 121, 204 and 267 have been preserved at least from the echinoderm to the vertebrates. A similar analysis has been done by Davidson. An intron at codon 150 is common to a plant actin gene and the skeletal muscle acting gene.