Association of small ankyrin 1 with the sarcoplasmic reticulum

Small ankyrin 1, or sAnk1, is a small, alternatively spliced product of the erythroid ankyrin gene, ANK1, that is expressed in striated muscle and concentrated in the network sarcoplasmic reticulum (SR) surrounding the Z disks and M lines. We have characterized sAnk1 in muscle homogenates and SR vesicles, and have identified the region that targets it to the network SR. Selective extractions and partitioning into Triton X-114 show that sAnk1 behaves like the SR Ca-ATPase and so is an integral protein of the SR membrane. Mild proteolytic treatment of isolated SR vesicles indicates that sAnk1 is oriented with its hydrophilic, C-terminal sequence exposed to the solution, which is equivalent to the cytoplasmic face of the SR membrane in situ. SDS-PAGE in non-reducing gels suggests that sAnk1 is present as dimers and larger oligomers in the native SR. These results suggest that sAnk1 is oligomeric and oriented with its C-terminus exposed to the cytoplasm, where it may interact with proteins of the contractile apparatus. The N-terminal 29 amino acid hydrophobic sequence of sAnk1, which is predicted to span the SR membrane, is sufficient to target proteins to and anchor them in internal membranes of HEK 293 cells. It also targets reporter proteins to the network SR of skeletal myofibers and is thus the first example of a sequence that targets proteins to a particular compartment of the SR.     

Obscurin is a ligand for small ankyrin 1 in skeletal muscle

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.     

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ∼2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca²⁺ affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca²⁺ in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca²⁺ misregulation.     

Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.     

The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.     

Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5). Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk-associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.     

Mapping the binding site on small ankyrin 1 for obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.     

Cytoplasmic gamma-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

The sarcoplasmic reticulum (SR) serves as the Ca(2+) reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γ(cyto)-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γ(cyto)-actin and Tmod3 defined an SR microdomain that was distinct from another Z line-flanking SR microdomain containing Tmod1 and Tmod4. The γ(cyto)-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γ(cyto)-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3-γ(cyto)-actin-sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca(2+) release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γ(cyto)-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.     

Effect of post-mortem electrical stimulation on ovine sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum (SR) vesicles from ovine skeletal muscle were iodinated with the use of immobilized lactoperoxidase to determine the location of proteins in the membrane and to observe any changes resulting from post-mortem electrical stimulation. The labelling pattern of the non-stimulated SR preparations was esentially the same as that observed previously for white muscle SR of rabbit. Most of the membrane protein were labelled, except for the high-affinity calcium-binding protein. Electrical stimulation, however, resulted in an increased labelling of calsequestrin suggesting that this protein is more exposed as a result of such treatment. Certain activities of the adenosinetriphosphatase were affected by electrical stimulation. Both the steady-state concentration of phosphoenzyme and the ATP in equilibrium Pi exchange reaction were significantly reduced by electrical stimulation. It is not known if this alteration in the membrane is responsible for the reduced activity of the SR and thus the greater rate of post-mortem pH in electrically stimulated muscle.     

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.     

Characterization and comparison of two binding sites on obscurin for small ankyrin 1

Obscurin A, an ～720 kDa modular protein of striated muscles, binds to small ankyrin 1 (sAnk1, Ank 1.5), an integral protein of the sarcoplasmic reticulum, through two distinct carboxy-terminal sequences, Obsc(6316-6436) and Obsc(6236-6260). We hypothesized that these sequences differ in affinity but that they compete for the same binding site on sAnk1. We show that the sequence within Obsc(6316-6436) that binds to sAnk1 is limited to residues 6316-6345. Comparison of Obsc(6231-6260) to Obsc(6316-6345) reveals that Obsc(6316-6345) binds sAnk1 with an affinity (133 ± 43 nM) comparable to that of the Obsc(6316-6436) fusion protein, whereas Obsc(6231-6260) binds with lower affinity (384 ± 53 nM). Oligopeptides of each sequence compete for binding with both sites at half-maximal inhibitory concentrations consistent with the affinities measured directly. Five of six site-directed mutants of sAnk1 showed similar reductions in binding to each binding site on obscurin, suggesting that they dock to many of the same residues of sAnk1. Circular dichroism (CD) analysis of the synthetic oligopeptides revealed a 2-fold greater α-helical content in Obsc(6316-6346), ～35%, than Obsc(6231-6260,) ～17%. Using these data, structural prediction algorithms, and homology modeling, we predict that Obsc(6316-6345) contains a bent α-helix of 12 amino acids, flanked by short disordered regions, and that Obsc(6231-6260) has a short, N-terminal α-helix of 4-5 residues followed by a long disordered region. Our results are consistent with a model in which both sequences of obscurin differ significantly in structure but bind to the ankyrin-like repeat motifs of sAnk1 in a similar though not identical manner.     

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease

A unique set of high molecular weight proteins was identified in junctional sarcoplasmic reticulum (SR) vesicles isolated from both cardiac muscle and skeletal muscle. These high Mr proteins were not present in free SR vesicles isolated from either tissue, nor were they observed in purified sarcolemmal fractions. The junctional SR high Mr proteins migrated as doublets in sodium dodecyl sulfate-polyacrylamide gels and exhibited apparent Mr values between 290,000 and 350,000. The high Mr proteins bound calmodulin; they were the principal proteins labeled in the cardiac and skeletal muscle SR subfractions by azido-125I-calmodulin. The high Mr proteins were also substrates for an endogenous Ca2+-calmodulin-dependent protein kinase activity, as well as exogenously added catalytic subunit of cAMP-dependent protein kinase. In addition, the junctional SR high Mr proteins were the major SR proteins degraded by a Ca2+-activated protease purified from smooth muscle. Control experiments verified the separation of junctional SR vesicles and free SR vesicles from both muscle types. Junctional SR vesicles were enriched in calsequestrin, and they exhibited Ca2+ uptake which was stimulated up to 10-fold by either ryanodine or ruthenium red. Free SR vesicles were deficient in calsequestrin and were insensitive to these two agents. Localization of the cardiac and skeletal muscle high Mr proteins to the junctional SR, coupled with demonstration of their nearly identical biochemical properties, suggests that the proteins are homologous and are likely to have similar functions in both types of striated muscle.     

Molecular evolution of ankyrin: gain of function in vertebrates by acquisition of an obscurin/titin-binding-related domain

Ankyrins form a family of modular adaptor proteins that link between integral membrane proteins and the cytoskeleton. They evolved within the Metazoa as an adaptation for organizing membrane microstructure and directing membrane traffic. Molecular cloning has identified one Caenorhabditis elegans (unc-44), two Drosophila (Dank1, Dank2), and three mammalian (Ank1, Ank2, Ank3) genes. We have previously identified a 76-amino acid (aa) alternatively spliced sequence that is present in muscle polypeptides encoded by the rat Ank3 gene. A closely related sequence in a muscle Ank1 product binds the cytoskeletal muscle proteins obscurin and titin. This obscurin/titin-binding-related domain (OTBD) contains repeated modules of 18 aa: three are encoded by Ank1 and Ank2, two by Ank3; this pattern is conserved throughout vertebrate ankyrin genes. The C. elegans ankyrin, UNC-44, contains one 18-aa module as does the ankyrin gene in the urochordate Ciona intestinalis, but the insect ankyrins contain none. Our data indicate that an ancestral ankyrin acquired an 18-aa module which was preserved in the Ecdysozoa/deuterostome divide, but it was subsequently lost from arthropods. Successive duplications of the module led to a gain of function in vertebrates as it acquired obscurin/titin-binding activity. We suggest that the OTBD represents an adaptation of the cytoskeleton that confers muscle cells with resilience to the forces associated with vertebrate life.     

Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase facilitates the entry of alpha(1),beta(1)-Na(+)-K(+)-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type alpha(1)-Na(+)-K(+)-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of alpha(1)-Na(+)-K(+)-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization.     

Localization of the alpha 1 and alpha 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the dihydropyridine (DHP) receptor and ankyrin in rat skeletal muscle with immunofluorescence and immunogold labeling techniques. All three proteins were concentrated in the triad junction formed between the T-tubules and sarcoplasmic reticulum. The alpha 1 and alpha 2 subunits of the DHP receptor were colocalized in the junctional T-tubule membrane, supporting their proposed association in a functional complex and the possible participation of the alpha 2 subunit in excitation-contraction coupling. Ankyrin label in the triad showed a distribution different from that of the DHP receptor subunits. In addition, ankyrin was found in longitudinally oriented structures outside the triad. Thus, ankyrin might be involved in organizing the triad and in immobilizing integral membrane proteins in T-tubules and the sarcoplasmic reticulum.     

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

The interactions between mitochondria and sarcoplasmic reticulum and the proteome characterization of mitochondrion‐associated membrane from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in mitochondrion‐sarcoplasmic reticulum (SR) linking, a catalog of proteins from mitochondrion‐associated membrane (MAM) of New Zealand white rabbit skeletal muscle were analyzed by an optimized shotgun proteomic method. The membrane fractions were prepared by differential centrifugation and separated by 1D electrophoresis followed by a highly reproducible, automated LC‐MS/MS on the hybrid linear ion trap (LTQ)‐Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 459 proteins were identified from both of the two independent MAM preparations. Protein pI value, molecular weight range, and transmembrane region were calculated using bioinformatics softwares. One hundred one proteins were recognized as membrane proteins. This protein database suggested that the MAM preparations composed of proteins from mitochondrion, SR, and transverse‐tubule. This result indicated mitochondria physically linked with SR in rabbit skeletal muscle, voltage‐dependent anion channel 1 (VDAC1), VDAC2, and VDAC3 might participate in formation of the tethers between SR and mitochondria.     

Combined Low Calcium and Lack Magnesium Is a Risk Factor for Motor Deficit in Mice

To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca^{2 +} release from SR in the rabbit skeletal muscle treated with high pressure (100–300 MPa, 5min) were investigated in comparison with those of the SR from conditioned muscle.

The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS–PAGE profile were not observed in the SR from the pressurized muscle up to 200 MPa, but a marked decrease of the ATPase protein and high-affinity Ca²⁺-binding protein were observed in the SR from the pressurized muscle at 300 MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200 MPa. When the muscle was pressurized at 300 MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle.Ca²⁺ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca²⁺, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle.

It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning.

The pressure-induced Ca^{2 +} release and loss of the structural regularity of myofibrils may be one of the causes for meat tenderization and acceleration of meat conditioning induced by high-pressure treatment.     

Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.