Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Glycosphingolipids of skeletal muscle: II. Modulation of Ca2(+)-flux in triad membranes by gangliosides.

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr, Res., (1988) 135-145). GM3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca(2+)-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca(2+)-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. GM3(Neu5Ac) strongly increased the uptake of 45Ca2+ (+285%) whereas GM3(Neu5Gc) severely inhibited the ion flux (-61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca(2+)-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI3Neu5Ac-nLcOse6Cer (C24-fatty acid), IV3Neu5Ac-nLcOse4Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C16-fatty acid) strongly enhanced the 45Ca(2+)-flux (+208, +162, and +120%, respectively, whereas IV3Neu5Ac-nLcOse4Cer (C24-fatty acid), VI3Neu5Ac-nLcOse6Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C24-fatty acid) slightly reduced 45Ca(2+)-flux (-3, -6, and -17%, respectively). Out of all gangliosides tested in this study, GM1 showed the strongest stimulatory effect (+327%). GD1a and GT1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas GD1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

Purification of the CIC-0 chloride channel from Torpedo california electroplax identification of a phosphorylation site for cAMP-dependent protein kinase.

The voltage-gated chloride channel (CIC-0) from the electric organ of Torpedo californica was purified by immunoaffinity chromatography. A polyclonal antibody was shown to specifically recognize the CIC-0 channel (M(r) 85,000-90,000) in a Western blot of total membrane proteins. As monitored by immunoprecipitation, the formation of antibody-antigen complexes in solution strongly depends on the detergent composition. The highest yield of precipitated CIC-0 was obtained from an incubation mixture containing both an anionic detergent, cholate or lauryl sulfate, and the zwitterionic detergent CHAPS. In contrast, immuno-precipitation of CIC-0 was largely reduced when cholate was exchanged for the nonionic detergent Triton x-100, suggesting that the efficient formation of immuno-complexes is favored by negatively charged detergent. In initial immunopurification experiments, in addition to CIC-0 a major contaminating polypeptide of M(r) approximately 115,000 was copurified, which represents the SITS-binding protein [Jentsch et al. (1989) Biochem. J. 261, 155]. Purification of CIC-0 could be increased from about 35% up to 60% homogeneity when immunoaffinity chromatography was performed in the presence of N-acetylglucosamine. Therefore the highly glycosylated SITS-binding protein most likely interacts with the CIC-0 protein via its carbohydrate parts. The purified CIC-0 channel was found to be phosphorylated by PKA in vitro to a level of 0.35-0.4 mol of phosphate incorporated per mol of CIC-0. Proteolytic digestion with endoproteinase GluC and HPLC-separation revealed two major phosphopeptides, which could be identified by amino acid sequence analysis as different size fragments of the same consensus phosphorylation site. Comparison of the peptide sequences with the deduced protein sequence of CIC-0 [Jentsch et al. (1990) Nature 348, 510; O'Neill et al. (1991) Biochem. Biophys. Acta 1129, 131] indicates serine 600 as the phosphorylated residue. Therefore, our results provide strong evidence that CIC-0 is phosphorylated at this single site by PKA in vitro.

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases.

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, GM3(Neu5Ac), GM3(Neu5Gc) and Gm1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was GM3(Neu5Ac) followed by moderately expressed GM3(Neu5Gc) and GM1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-GM3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle.

Sarcolemmal chloride and potassium channels from normal and myotonic mouse muscle studied in lipid supplemented vesicles.

By patch-clamp analysis of lipid supplemented vesicles prepared from the sarcolemma of mouse wildtype skeletal muscle, we could identify two known types of potassium channels, the inward rectifier and Ca2+ activated BK channels, and five types of chloride channels designated CIC-a, CIC-b, CIC-c, CIC-d, and CIC-e. CIC-b corresponds to a known chloride channel, whereas CICa, -c, -d and -e have not been described previously. The diversity may be due to a heterooligomeric composition of different subunits. None of these chloride channels nor the potassium channels were found in the sarcoplasmic reticulum fraction. In vesicles from myotonic mice, Clc-d and -e were not found.

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining.

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neural glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides GM3(Neu5Ac) and GM1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphingolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of GM3 and GM1 correlated to the fact that GM3 is the major ganglioside in skeletal muscle whereas GM1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. GM3 and GM1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level.

Different distributions of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses.

The expression of neutral glycosphingolipids and gangliosides was investigated in mouse and rabbit skeletal muscle by means of biochemical and immunochemical techniques. Neutral glycosphingolipids from muscle of the inbred rabbit strain used in this study showed a simple TLC pattern, comprising mainly monohexosylceramide. In addition to this compound, lactosylceramide, lacto-N-neotetraosylceramide, globoside and Forssman GSL were detected in mouse muscle. The major ganglioside in both species was GM3; GM3 (Neu5Ac) and GM3(Neu5Gc) were found in a 3:1 ratio in mouse muscle, whereas the absence of GM3(Neu5Gc) is characteristic of rabbit muscle. As a general structural feature of all muscle GM3 gangliosides investigated, a C18 fatty acid and C18 sphingosine were the major components besides minor C22 and C24:1 fatty acids of the respective ceramide portions, as revealed by positive and negative ion FAB-MS. alpha 2-3 sialylated lacto-N-neotetraosyl-ceramide (sialylparagloboside) was expressed in both species, whereas the alpha 2-6 sialylated isomeric compound was found only in mouse muscle. Minute quantities of ganglio-series GM1, GD1a, GD1b, and GT1b were detected in muscles from both species. Glycosphingolipid expression could be confirmed immunohistochemically by examining transverse and longitudinal cryosections of skeletal muscle samples. The results provide the basis for the investigation of muscle specific glycosphingolipids that might modulate membrane protein functions in muscle.

Indanyloxyacetic acid-sensitive chloride channels from outer membranes of skeletal muscle.

In mature mammalian muscle, the chloride conductance of the membrane is an important factor in the regulation of excitability. Up to now, no ligand was available for the biochemical characterization of muscle chloride channels. In order to localize and characterize these channels, we have used indanyloxyacetic acid (IAA)-94, a ligand previously used for epithelial Cl- channels (Landry, D. W., Reitman, M., Cragoe, E. J., Jr., and Al-Awqati, Q. (1987) J. Gen. Physiol. 90, 779-798; Landry, D. W., Akabas, M. H., Redhead, C., Edelman, A., Cragoe, E. J., Jr., and Al-Awqati, Q. (1989) Science 244, 1469-1472). IAA induced myotonic responses when microinjected into mature mouse muscle fibers, indicating a blockade of Cl- channels from the cytoplasmic side. Membrane vesicles were prepared from rabbit skeletal muscle and separated by sucrose gradient centrifugation. Fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmatic reticulum (SR), and triads and mitochondria (TR/M). The fraction enriched for SL was characterized by high specific binding capacity for [3H]saxitoxin (Na+ channel), whereas TT-rich fractions bound [3H]PN 200-110 (dihydropyridine receptor) with high specific activity. Upon patch-clamping of lipid supplemented vesicles, IAA-sensitive Cl- channels were found in the SL fraction but not in the SR. Highest specific activities in electrical diffusion potential sensitive 36Cl transport and [3H]IAA-94 binding were found in the SL. SL vesicles were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate and subjected to IAA-Sepharose affinity chromatography. Specifically bound protein was eluted with 100 microM IAA-94 and either analyzed by SDS-gel electrophoresis or reconstituted into phospholipid vesicles. The eluate contained four polypeptides (specifically bound, mapp 110-120 and 60 kDa; unspecifically bound mapp 67 and 50 kDa) and was highly enriched for IAA-sensitive chloride channels as shown by patch-clamping after reconstitution. The IAA-sensitive 100/280-picosiemens chloride channels of the sarcolemma are likely to be responsible for its major chloride conductance and thereby for the stabilization of resting potential.