Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Simvastatin triggers mitochondria-induced Ca2+ signaling alteration in skeletal muscle

Statin drugs represent the major improvement in the treatment of hypercholesterolemia that constitutes the main origin of atherosclerosis, leading to coronary heart disease. Besides tremendous beneficial effects of statins, various forms of muscular toxicity (myalgia, cramp, exercise intolerance, and fatigability) occur frequently. We hypothesized that the iatrogenic effects of statins could result from alterations in Ca²⁺ homeostasis. Acute applications of simvastatin on human skeletal muscle fibers triggered a Ca²⁺ wave of intra-cellular Ca²⁺ that mostly originates from sarcoplasmic reticulum (SR) Ca²⁺-release. In addition, simvastatin increased mitochondrial NADH content and induced mitochondrial membrane depolarization (EC₅₀ = 1.96 μM) suggesting an altered mitochondrial function. Consequently on simvastatin application, a weak mitochondrial Ca²⁺ efflux (EC₅₀ = 7.8μM) through permeability transient pore and Na⁺/Ca²⁺ exchanger was triggered, preceding the large SR-Ca²⁺ release. Increased SR Ca²⁺ content after acute application of statin is also suggested by the increased Ca²⁺ spark amplitude and by the effect of cyclopiazonic acid. We thus conclude that simvastatin induced alterations in mitochondrial function which lead to an increase in cytoplasmic Ca²⁺, SR-Ca²⁺ overload, and Ca²⁺ waves. Taken together, these statin-induced muscle dysregulations may contribute to myotoxicity.     

The gating of the sheep skeletal sarcoplasmic reticulum Ca2+-release channel is regulated by luminal Ca2+

The effects of changes in luminal [Ca²⁺] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca²⁺-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mm Cs⁺. Lifetime analysis indicates that channels activated solely by cytosolic Ca²⁺ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca²⁺, luminal Ca²⁺ does not appear to exert any regulatory effect. The P ₀ and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca²⁺, increases in luminal [Ca²⁺] produce marked increases in P ₀ and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca²⁺-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca²⁺].We are grateful to the British Heart Foundation for financial support.     

Ca channels and skeletal muscle diseases

When observed under a microscope, skeletal muscle exhibits striations due to the highly organized arrangement of muscle proteins that interact with one another to induce muscle contraction. Muscle contraction requires transient increases in intracellular ‘Ca²⁺’ concentration. In this review, Ca²⁺ channels contributing to the functional integrity of intracellular Ca²⁺-release and extracellular Ca²⁺-entry during skeletal muscle contraction are reviewed in terms of their properties, newly emerging ancillary proteins to them, and their abnormalities related to human skeletal muscle diseases. Finally, the aim of this review is to show the big picture of the correlation among Ca²⁺ channels that participate in the Ca²⁺ homeostasis in skeletal muscle.     

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.     

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Androgen receptor antagonist suppresses exercise-induced hypertrophy of skeletal muscle

The physiological importance of the increase in androgen receptors in exercise-induced muscle hypertrophy was investigated in rats. Together with training rat gastrocnemius muscles by electrical stimulation every other day for 2 weeks, male rats were administered the androgen receptor antagonist, oxendolone. The androgen receptor antagonist effectively decreased the wet mass of the prostate, an androgen target organ, and did not significantly affect body mass. The increase in muscle mass induced by electrical stimulation was effectively suppressed by the androgen receptor blockade. The mean degree of muscle hypertrophy in the antagonist-treated group was significantly lower than that in the control group (102.30% vs 107.41%, respectively;P=0.006). This result suggests that the androgen pathway has a significant effect in exercise-induced muscle hypertrophy and emphasizes the importance of the increase in the number of androgen receptors in exercised muscle.     

Ca2+ sparks in embryonic mouse skeletal muscle selectively deficient in dihydropyridine receptor alpha1S or beta1a subunits.

Ca2+ sparks are miniature Ca2+ release events from the sarcoplasmic reticulum of muscle cells. We examined the kinetics of Ca2+ sparks in excitation-contraction uncoupled myotubes from mouse embryos lacking the beta1 subunit and mdg embryos lacking the alpha1S subunit of the dihydropyridine receptor. Ca2+ sparks occurred spontaneously without a preferential location in the myotube. Ca2+ sparks had a broad distribution of spatial and temporal dimensions with means much larger than those reported in adult muscle. In normal myotubes (n = 248 sparks), the peak fluorescence ratio, DeltaF/Fo, was 1.6 +/- 0.6 (mean +/- SD), the full spatial width at half-maximal fluorescence (FWHM) was 3.6 +/- 1.1 micrometer and the full duration of individual sparks, Deltat, was 145 +/- 64 ms. In beta-null myotubes (n = 284 sparks), DeltaF/Fo = 1.9 +/- 0.4, FWHM = 5.1 +/- 1.5 micrometer, and Deltat = 168 +/- 43 ms. In mdg myotubes (n = 426 sparks), DeltaF/Fo = 1 +/- 0.5, the FWHM = 2.5 +/- 1.1 micrometer, and Deltat = 97 +/- 50 ms. Thus, Ca2+ sparks in mdg myotubes were significantly dimmer, smaller, and briefer than Ca2+ sparks in normal or beta-deficient myotubes. In all cell types, the frequency of sparks, DeltaF/Fo, and FWHM were gradually decreased by tetracaine and increased by caffeine. Both results confirmed that Ca2+ sparks of resting embryonic muscle originated from spontaneous openings of ryanodine receptor channels. We conclude that dihydropyridine receptor alpha1S and beta1 subunits participate in the control of Ca2+ sparks in embryonic skeletal muscle. However, excitation-contraction coupling is not essential for Ca2+ spark formation in these cells.     

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle-tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle-tendon junction, tendon, tendon-bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.     

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Ca2+ effects on glucose transport and fatty acid oxidation in L6 skeletal muscle cell cultures

We examined the effect of Ca²⁺ on skeletal muscle glucose transport and fatty acid oxidation using L6 cell cultures. Ca²⁺ stimulation of glucose transport is controversial. We found that caffeine (a Ca²⁺ secretagogue) stimulation of glucose transport was only evident in a two-part incubation protocol (“post-incubation”). Caffeine was present in the first incubation, the media removed, and labeled glucose added for the second. Caffeine elicited a rise in Ca²⁺ in the first incubation that was dissipated by the second. This post-incubation procedure was insensitive to glucose concentrations in the first incubation. With a single, direct incubation system (all components present together) caffeine caused a slight inhibition of glucose transport. This was likely due to caffeine induced inhibition of phosphatidylinositol 3-kinase (PI3K), since nanomolar concentrations of wortmannin, a selective PI3K inhibitor, also inhibited glucose transport, and previous investigators have also found this action.We did find a Ca²⁺ stimulation (using either caffeine or ionomycin) of fatty acid oxidation. This was observed in the absence (but not the presence) of added glucose. We conclude that Ca²⁺ stimulates fatty acid oxidation at a mitochondrial site, secondary to malonyl CoA inhibition (represented by the presence of glucose in our experiments). In summary, the experiments resolve a controversy on Ca²⁺ stimulation of glucose transport by skeletal muscle, introduce an important experimental consideration for the measurement of glucose transport, and uncover a new site of action for Ca²⁺ stimulation of fatty acid oxidation.     

Acetylcholine receptor subunit expression in Huntington's disease mouse muscle

Huntington's disease (HD) causes neurological impairments, as well as muscle dysfunction, including smaller neuromuscular junctions (NMJs). This study assessed the expression levels of the subunits of the nicotinic acetylcholine receptor (nAChR) in muscles of the R6/2 mouse model of HD. Based on our previous findings of reduced NMJ size in R6/2 mice, it was hypothesized that muscles from R6/2 mice would also show an altered expression pattern of nAChR subunits compared to wild-type (WT) mice. Therefore, the mRNA levels of nAChR subunits were quantified in R6/2 and WT mouse muscles using qRT-PCR. Denervated muscles from WT mice served as positive controls for alterations in nAChR expression. Although some changes in nAChR subunit expression occurred in R6/2 muscles, the expression levels closely resembled WT. However, the expression of nAChR subunit-ε (Chrne) was significantly decreased in R6/2 muscles relative to WT. This study demonstrates that only minor changes in nAChR subunit expression occurs in R6/2 mouse muscles and that reduction in Chrne expression may be related to a reduction in NMJ size in R6/mice.     

3D structure of the skeletal muscle dihydropyridine receptor

The dihydropyridine receptors (DHPR) are L-type voltage-gated calcium channels that regulate the flux of calcium ions across the cell membrane. Here we present the three-dimensional (3D) structure at approximately 27A resolution of purified skeletal muscle DHPR, as determined by electron microscopy and single particle analysis. Here both biochemical and 3D structural data indicate that DHPR is dimeric. DHPR dimers are composed of two arch-shaped monomers approximately 210A across and approximately 75A thick, that interact very tightly at each end of the arch. The roughly toroidal structure of the two monomers encloses a cylindrical space of approximately 80A diameter, which is then closed on each side by two dome-shaped protein densities reaching over from each monomer arch. The dome-shaped domains have a length of approximately 80-90A and a maximum height of approximately 45A. Small orifices punctuate their exterior surface. The 3D structure disclosed here may have important implications for the understanding of DHPR Ca(2+) channel function. We also propose a model for its in vivo interactions with the calcium release channel at the junctional sarcoplasmic recticulum.     

Compromised store-operated Ca2+ entry in aged skeletal muscle

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca²⁺ entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca²⁺ entry (SOCE) in aged mouse skeletal muscle. Using Mn²⁺ quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca²⁺ movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26–27 months) as compared with those from young (2–5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca²⁺ homeostatic capacity generally associated with muscle aging.     

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.     

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle

Background

δ-Sarcoglycan (δ-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca²⁺ permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease.

Methods

Isolated skeletal muscle fibers of wild-type and δ-SG KO mice were used to investigate whether the absence of δ-SG alters the increase in intracellular Ca²⁺ during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca²⁺ transient recording techniques with fluorescent indicators were used.

Results

Ca²⁺ transients during single twitches and tetani generated by muscle fibers of δ-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca²⁺ and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a δ-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles.

Conclusions

δ-SG has a role in calcium homeostasis in skeletal muscle fibers.

General significance

These results support a possible role of δ-SG on calcium homeostasis. The alterations caused by the absence of δ-SG may be related to the pathogenesis of muscular dystrophy.     

Sarcoplasmic Reticulum Ca2+ Release in Rat Slow- and Fast-Twitch Muscles

The same isoform of ryanodine receptor (RYR1) is expressed in both fast and slow mammalian skeletal muscles. However, differences in contractile activation and calcium release kinetics in intact and skinned fibers have been reported. In this work, intracellular Ca²⁺ transients were measured in soleus and extensor digitorum longus (EDL) single muscle fibers using mag-fura-2 (K _D for Ca²⁺= 49 μm) as Ca²⁺ fluorescent indicator. Fibers were voltage-clamped at V _h =−90 mV and sarcoplasmic reticulum calcium release was measured at the peak (a) and at the end (b) of 200 msec pulses at +10 mV. Values of a-b and b were assumed to correspond to Ca²⁺-gated and voltage-gated Ca²⁺ release, respectively. Ratios (b/a-b) in soleus and EDL fibers were 0.41 ± 0.05 and 1.01 ± 0.13 (n= 12), respectively. This result suggested that the proportion of dihydropyridine receptor (DHPR)-linked and unlinked RYRs is different in soleus and EDL muscle. The number of DHPR and RYR were determined by measuring high-affinity [³H]PN200-110 and [³H]ryanodine binding in soleus and EDL rat muscle homogenates. The B _max values corresponded to a PN200-110/ryanodine binding ratio of 0.34 ± 0.05 and 0.92 ± 0.11 for soleus and EDL muscles (n= 4–8), respectively. These data suggest that soleus muscle has a larger calcium-gated calcium release component and a larger proportion of DHPR-unlinked RYRs. Received: 31 August 1995/Revised: 25 January 1996