Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling

Summary Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the ₂, and subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.     

Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres

Summary The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles ofXenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 m to 150 m and those with diameters > 10 m are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 m. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca²⁺ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctionalface membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).     

The fenestrated collar of mammalian cardiac sarcoplasmic reticulum: A freeze-fracture study

Freeze-fracture studies of papillary muscles from cat, rabbit and dog reveal the presence of a fenestrated collar of the sarcoplasmic reticulum (SR) in the region of the M band. This membrane specialization is structurally similar to that observed previously in skeletal muscle. This report includes mammalian cardiac muscle on the list of those muscles containing this SR membrane structure.     

Beta-amyloid protein-containing inclusions in skeletal muscle of apolipoprotein-E-deficient mice.

The tibialis anterior muscle and soleus muscle of apolipoprotein-E-deficient mice were examined by light and electron microscopy. By light microscopy, sarcoplasmic inclusions were seen in tibialis anterior muscle and 40% of type 2 myofibers were affected in all animals over 8 months of age. These inclusions reacted for nonspecific esterase, cytochrome oxidase, and myoadenylate deaminase and were also periodic acid Schiff positive and stained basophilic with hematoxylin. Moreover, they reacted immunocytochemically with an antibody specific to fragment 17 to 24 of the published sequence of Alzheimer's cerebrovascular amyloid peptide. Immunoreactivity was lost when the antibody was adsorbed with the appropriate synthetic peptide. Ultrastructurally, the inclusions consisted of tubular arrays and were similar to those observed in human muscle in several pathological conditions. In type 1 myofibers of both tibialis anterior and soleus muscle, however, mitochondrial abnormalities including an increase in their number and size were detected, but tubular aggregates were not seen. These large mitochondria possessed an electron-dense inner chamber with an increased number of tightly packed cristae. The results obtained suggest that in these mice there is a disturbed lipid metabolism in skeletal muscle fibers that manifests itself with an accumulation of phospholipid in the form of sarcoplasmic reticulum tubules in the type 2 fibers and enlarged mitochondria with tightly packed cristae in the type 1 fibers. In addition, beta-amyloid protein was closely associated with the accumulated tubules and vesicles of sarcoplasmic reticulum and may represent dysregulation of amyloid precursor protein metabolism.     

Ultra-high resolution scanning electron microscopic studies on the sarcoplasmic reticulum and mitochondria in the rat cardiac and skeletal muscle fibers

The three-dimensional structure of the sarcoplasmic reticulum (SR), transverse (T)-axial tubular system and mitochondria in cardiac and skeletal muscle fibers of the rat was examined by ultra-high resolution scanning electron microscopy after removal of the cytoplasmic matrices and myofilaments by the aldehyde-osmium-dimethyl sulfoxide-osmium procedure^1,2. Between the cardiac and the skeletal muscle fibers, striking differences in the three-dimensional structure of the mitochondria and of the SR were observed.     

Distribution of ryanodine receptors in rat ventricular myocytes

Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca²⁺ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca²⁺ cycling and Ca²⁺-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.     

Ultrastructural localization of calcium in normal and abnormal skeletal muscle.

Calcium was demonstrated ultrastructurally as a fine black reaction product with unbuffered 2% saturated potassium pyroantimonate, pH 9.4. In comparison with normal muscle, there was increased precipitate in degenerating skeletal muscle fibers and some degenerating-regenerating fibers occurring in pathologic human muscle, regardless of disease entity, and in experimentally injured rat muscle. The pathologically increased calcium was mainly within the sarcoplasmic reticulum and mitochondria. Both structures could be completely blackened. Nuclear calcium was also increased, the precipitates being localized as circular profiles within the nucleoli and heterochromatin as well as being associated with the nuclear envelope. Myofibrillar calcium was only modestly increased. When normal rat muscle was preincubated in 136 mM calcium-enhanced Hanks' medium, calcium accumulated in the muscle fibers--it was especially heavy in the mitochondria and sarcoplasmic reticulum and appeared identical with the pathologic human and rat muscle fibers. Preincubation of normal rat muscle in 0.1 M acetate buffer (pH 4.65) before calcium loading augmented myofibrillar staining, mainly in the H-zone of the A-bands excluding the M-zone and in broad irregular N1, N2, and "N3" lines of the I-bands. EMMA-4 electron probe microanalysis and EGTA (ethylene glycolbis (beta-aminoethyl ether)N,N'-tetraacetic acid) chelation prior to staining confirmed that the precipitate in the several loci was calcium antimonate. It is proposed that in skeletal muscle fibers injured by various pathologic processes, a breach of the plasmalemma barrier to calcium occurs as a very early abnormality. Extracellular calcium would then pour into the aqueous sarcoplasm of the muscle fiber, from which it would be withdrawn by and accumulated with the still active organelles normally having a great avidity for uptake of this ion, especially the mitochondria and sarcoplasmic reticulum. The resultant organellar calcification would impair function and damage the structure of proteins and phospholipids.     

Distribution of (Na+ + K+)ATPase and sodium channels in skeletal muscle and electroplax

Summary The distributions of (Na⁺ + K⁺)ATPase and sodium channels in skeletal muscle fibres and electrocytes were determined by immunofluorescent and immunoelectron microscopic techniques using antibodies against rat and eel (Na⁺ + K⁺)ATPase and the eel electric organ sodium channel. The extrajunctional sarcolemma of skeletal muscle was uniformly stained by polyclonal antibodies against (Na⁺ + K⁺)ATPase and the sodium channel. The T-tubule system of skeletal muscle was also labelled heavily for both (Na⁺ + K⁺)ATPase and the sodium channel. The terminal cisternae of the sarcoplasmic reticulum was stained for (Na⁺ + K⁺)ATPase but not sodium channels. At the motor endplate, (Na⁺ + K⁺)ATPase-like immunoreactivity was present along the plasmalemma of motor nerve terminals but not along the postsynaptic junctional sarcolemma. Paradoxically, a monoclonal antibody that binds to the form of the catalytic subunit of (Na⁺ + K⁺)ATPase from rat hepatocytes and renal tubule cells did not label the enzyme in rat skeletal muscle. In electrocytes, (Na⁺ + K⁺)ATPase-like irnmunoreactivity was concentrated primarily along the plasmalemma and calveolae of the non-innervated face. In contrast, sodium channel-like immunoreactivity was concentrated along the plasmalemma of the innervated face except in the clefts of the postsynaptic membrane. Thus, we conclude that at endplates both the (Na⁺ + K⁺)ATPase of rat skeletal muscle and sodium channels of eel electrocytes are not concentrated in the juxtaneuronal postsynaptic membrane. We also interpret the failure of the monoclonal anti- (Na⁺ + K⁺)ATPase antibodies to bind to the enzyme in muscle to indicate that the catalytic subunit of skeletal muscle (Na⁺ + K⁺)ATPase displays different epitopes than does the a subunit of kidney and liver.     

Possible role of cytoskeleton in intracellular arrangement and regulation of mitochondria

The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent K(m) for exogenous ADP (250-350 mM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 mm) is independent of intrinsic MgATPase activity. However, the K(m) may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum.     

Morphometry of myocardial apex in endurance-trained mice of different ages

Mitochondrial volume density, surface density of the outer mitochondrial membrane, the mean number and size of mitochondria, and the mean surface density of crista membranes together with the volume densities of myofibrils and sarcoplasmic space were morphometrically analyzed in cardiac muscle of two groups of sedentary control mice aged 3 and 7 months, and in two groups of mice trained either 1 month rather intensely or 4 months moderately. Of the calculated mitochondrial variables only the surface density of the outer mitochondrial membrane differed between the older controls and the older trained animals, the density being slightly smaller in the trained group. The myofibrillar volume density of the order controls was smaller than that of the younger controls, while the sarcoplasmic volume density was larger. The latter difference, possibly a function of age, was also observed in the trained groups. The results suggest that at a certain steady state level of exercise-induced cardiac muscle hypertrophy the muscle cells of trained mice do not differ markedly in ultrastructural properties from those of sedentary controls.     

Control of intracellular Ca2+ activity in rat myometrium.

Four fractions enriched, respectively, in plasma membrane (PM), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria were isolated from estrogen-dominated rat myometrium. Ca2+ uptake by these fractions was studied in order to estimate the relative potential of the corresponding organelles for controlling intracellular Ca2+ activity. Ca2+ uptake properties of the PM, SER, and RER fractions were similar except that potentiation by oxalate was in the order RER greater than or equal SER greater than PM. However, studies with the ionophores X-537A and A23187 suggested that Ca2+ was transported into the lumen of membrane vesicles of all these fractions. Unlike that of skeletal muscle sarcoplasmic reticulum, Ca2+ uptake by the myometrial fractions was not supported by high-energy compounds other than ATP. Mitochondria took up much less Ca2+ at low, and much more Ca2+ at high, free Ca2+ concentrations than did the other fractions. The amount of Ca2+ taken up in 30 s from a 1 muM free Ca2+ solution in the presence of ATP was similar for all fractions. These results suggested that mitochondria may act as an important Ca2+ control system in rat myometrium when the intracellular Ca2+ concentration is near 1 muM or higher, whereas the PM, SER, and RER may be of major importance at Ca2+ levels of 0.3 muM or lower.     

Calsequestrin expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle though not in cardiac muscle

Altered mechanisms of Ca2+ transport may underlie the contractile dysfunctions that have been frequently reported to occur in diabetic cardiac and skeletal muscle tissues. Calsequestrin, a high-capacity Ca2+-binding protein, is involved in the regulation of the excitation-contraction-relaxation cycle of both skeletal and cardiac muscle fibres. We have investigated the expression of calsequestrin and Ca2+ binding in cardiac and skeletal muscle from streptozotocin-induced diabetic rat. Immunoblotting of microsomal membranes from normal and streptozotocin-induced diabetic muscle revealed no significant changes in heart, but an increase in the relative abundance of calsequestrin and calsequestrin-like proteins in skeletal muscle. In analogy, the overall Ca2+-binding capacity of sarcoplasmic reticulum vesicles from diabetic skeletal muscle was drastically increased. The expression of fast muscle marker proteins was not affected, indicating that no relevant fibre transformation occurred in streptozotocin-treated rat muscles. The up-regulation of the high-capacity Ca2+-binding element calsequestrin might represent a compensatory mechanism of diabetic skeletal muscle. An increased Ca2+-buffering capacity of the sarcoplasmic reticulum lumen might counteract elevated cytosolic Ca2+ levels in diabetes thereby preventing Ca2+-dependent myo-necrosis.     

Localization by immunoelectron microscopy of spanning protein of triad junction in terminal cisternae/triad vesicles

Summary Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca²⁺ channel.Nomenclature SP spanning protein - SR sarcoplasmic reticulum - TC terminal cisternae - T-tubule transverse tubule - HMW high molecular weight - ELISA enzyme linked immunosorbent assay - PMSF phenylmethyl sulphonylfluoride - SDS sodium dodecyle sulphate     

Isolation, growth and differentiation of adult rabbit skeletal myoblasts in vitro

A simple and reliable method is described for the growth and differentiation of myoblasts isolated from adult New Zealand White rabbit fast-twitch (tibialis anterior) and slow-twitch (soleus) skeletal muscle. Cells were dissociated mechanically, and expanded in DMEM supplemented with 20% horse serum. The myoblasts were differentiated by switching to DMEM supplemented with 10% horse serum when the myoblasts were 80–90% confluent. The myoblasts fused into multinucleated myotubes that spontaneously contracted. At the light microscopic level, the myotubes exhibited a striated pattern as revealed by positive immunostaining for sarcomeric proteins such as -actinin, myosin and titin.Electron microscopy demonstrated well ordered sarcomeres with localized mitochondria and sarcoplasmic reticulum. The only notable difference between the fiber types was in the initial response to isolation wherein the myoblast yield was four-fold greater from the soleus than the tibialis anterior, in agreement with satellite cell abundance in predominately slow-twitch vs fast-twitch muscles. These techniques repeatedly (n = 12) produced a population of healthy myoblasts isolated from either fast- or slow-twitch skeletal muscle which can be utilized for studies of skeletal muscle differentiation, assembly and signaling.     

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 m s–1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen     

Histidine-rich calcium binding protein,a sarcoplasmic reticulum protein of striated muscle,is also abundant in arteriolar smooth muscle cells

Summary Histidine-rich calcium binding protein (HRC) is a luminal sarcoplasmic reticulum protein abundant in skeletal and cardiac muscle. Using immunofluorescence to examine non-muscle tissues, we now show that HRC is also abundant in the smooth muscle cells lining the walls of small arteries and arterioles. Arterioles that contain only one or two layers of smooth muscle cells are very brightly stained while small muscular arteries demonstrate a lesser degree of immunoreactivity only in cells just adjacent to the lumen of the vessel. In contrast, visceral smooth muscle cells from the gastrointestinal and genitourinary tracts show no HRC immunofluorescence. We also examined the subcellular distribution of HRC in arteriolar smooth muscle by immunoelectron microscopy. HRC was found in electron-dense vesicles beneath the plasma membrane, in small electron-lucent vesicles and in the nuclear envelope, suggesting a location within a calcium-sequestering compartment. These findings suggest that HRC plays a role in sarcoplasmic reticulum function that is unique to striated and arteriolar smooth muscle.     

Intracellular localization and isoform expression of the voltage-dependent anion channel (VDAC) in normal and dystrophic skeletal muscle

Voltage-dependent anion channels (VDACs) are a family of pore-forming proteins encoded by different genes, with at least three protein products expressed in mammalian tissues. The major recognized functional role of VDACs is to permit the almost free permeability of the outer mitochondrial membrane (OMM). Although VDAC1 is the best known among VDAC isoforms, its exclusively mitochondrial location is still debated. Therefore, we have measured its co-localization with markers of cellular organelles or compartments in skeletal muscle fibers by single or double immunofluorescence and traditional as well as confocal microscopy. Our results show that VDAC1 immunoreactivity corresponds to mitochondria and sarcoplasmic reticulum, while sarcolemmal reactivity, previously reported, was not observed. Since VDAC1 has been suggested to be involved in the control of oxidative phosphorylation, we sought for possible gene regulation of VDAC1, VDAC2 and VDAC3 in skeletal muscle of the dystrophin-deficient mdx mouse, which suffers of an impaired control of energy metabolism. Our results show that, while VDAC1 mRNA and protein and VDAC2 mRNA are normally expressed, VDAC3 mRNA is markedly down-regulated in mdx mouse muscle at different ages (before, during and after the outburst of myofiber necrosis). This finding suggests a possible involvement of VDAC3 expression in the early pathogenic events of the mdx muscular dystrophy.     

The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain

Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells.     

Cytochemical studies of a glycogen-sarcoplasmic reticulum complex

Summary Enzymatically active cardiac sarcoplasmic reticulum (SR) fractions contain glycogen. Previous biochemical and morphological studies indicate that the glycogen particles are membrane associated. In the present study, further evidence for membrane-associated glycogen particles in these cardiac SR fractions is presented: (1) morphological parameters, (2) enzymatic digestion by glucoamylase and alpha-amylase and (3) cytochemical staining by two different methods. Dense granules comparable in size (20–30 nm diameter), electron density and substructure to glycogen particles observed in intact cardiac muscle and in glycogen preparations isolated from skeletal muscle were seen. Most of these glycogen particles were removed by amylase digestion except for glycogen particles closely adhering to vesicle membranes. Two different cytochemical techniques (bismuth subnitrate and silver proteinate) revealed a positive reaction product over the glycogen particles. These findings provide further support for the biochemical finding of a structured enzyme complex involving the SR, glycogenolytic enzymes and glycogen.     

Actinlike material in Pseudomonas aeruginosa.

Actinlike material was obtained from disrupted Pseudomonas aeruginosa cells by a modification of the method of Hancock and Nikaido (J. Bacteriol. 136:381-390) for isolating outer membrane vesicles. Pelleted membranes were dissolved in Laemmli sample buffer and electrophoresed in parallel lanes with purified rabbit skeletal muscle actin. The bacterial preparation migrated similarly to rabbit skeletal muscle actin. A doublet band was detectable by affinity-purified antiactin antibody in a passive transfer immunoblot. Molecular weight of the actinlike protein doublet was 60,000 to 63,000 as determined by linear regression analysis of Bio-Rad low-molecular-weight standards run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transmission electron microscopy of the actinlike material in high salt concentrations revealed 10- to 14-nm filaments of various lengths. Despite its ability to form filaments and to react with a polyclonal rabbit antiactin antibody, the bacterial filaments did not bind the S-1 fragment of heavy meromyosin.