Co-localization of the dihydropyridine receptor and the cyclic AMP-binding subunit of an intrinsic protein kinase to the junctional membrane of the transverse tubules of skeletal muscle.

Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further distinguished from free TT by the presence of a high number (Bmax. 20 pmol/mg of protein) of [3H]cyclic AMP receptor sites, as determined by the Millipore filtration technique of Gill & Walton [(1974) Methods Enzymol. 38, 376-381]. Use of this method means that the number of receptors may have been underestimated. The TT-bound cyclic AMP receptor was identified as a 55 kDa protein by specific photoaffinity labelling with 8-N3-[3H]cyclic AMP, and had similar phosphorylation properties and apparent molecular mass to the RII form of the regulatory subunit of cyclic AMP-dependent protein kinase. Co-localization of the intrinsic cyclic AMP-dependent protein kinase and of the DHP receptor complex to the junctional membrane of TT supports the hypothesis that the 170 kDa alpha 1-subunit of the receptor is a substrate for the kinase.

Calsequestrin, an intracellular calcium-binding protein of skeletal muscle sarcoplasmic reticulum, is homologous to aspartactin, a putative laminin-binding protein of the extracellular matrix.

Calsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed.

Calcium-dependent phospholipid binding proteins associated with the membranes of rabbit skeletal muscle.

By using extraction in the presence of Ca2+ and Triton X-100 and then in the presence of EGTA without detergent, a set of Ca2+-dependent phospholipid binding proteins has been identified in the membranes of transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), isolated from rabbit skeletal muscles. Longitudinal SR, junctional SR and T-tubule membranes yielded about 9, 14 and 3.3 micrograms of EGTA-soluble proteins per 1 mg of membrane protein, respectively. In the presence of 1 mM CaCl2, 68 and 33 kDa proteins of T-tubules and junctional SR as well as 30 kDa protein of T-tubules were shown to bind to liposomes made of 1:1 w/w mixtures of (i) phosphatidylcholine and (ii) phosphatidylserine, phosphatidic acid, or phosphatidyl ethanolamine. In the presence of EGTA, the above-mentioned proteins were mostly found in the supernatants. Binding of the proteins with liposomes consisting of pure phosphatidylcholine was negligible.

The identification of sarcoplasmic reticulum terminal cisternae proteins in platelets.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences.

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.

Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers.

The behavior of single Cl- channel was studied by fusing isolated canine cardiac sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. The channel exhibited unitary conductance of 55 pS (in 260 mM Cl-) and steady-state activation. Subconductance states were observed. Open probability was dependent on holding potentials (-60 to +60 mV) and displayed a bell-shaped relationship, with probability values ranging from 0.2 to 0.8 with a maximum at -10 mV. Channel activity was irreversibly inhibited by DIDS, a stilbene derivative. Time analysis revealed the presence of one time constant for the full open state and three time constants for the closed states. The open and the longer closed time constants were found to be voltage dependent. The behavior of the channel was not affected by changing Ca2+ and Mg2+ concentrations in both chambers, nor by adding millimolar adenosine triphosphate, or by changing the pH from 7.4 to 6.8. The presence of sulfate anions decreased the unit current amplitude, but did not affect the open probability. These results reveal that at the unitary level the cardiac SR anion-selective channel has distinctive as well as similar electrical properties characteristic of other types of Cl- channels.

Albumin is a major protein component of transverse tubule vesicles isolated from skeletal muscle.

Despite multiple procedures used to isolate transverse tubule vesicles from rabbit skeletal muscle, few proteins have been identified and shown to be specific to transverse tubule vesicles. Markers for purified transverse tubules have included high affinity dihydropyridine binding, cholesterol content, Mg2+-ATPase activity, (Na+,K+)-ATPase activity, and [3H] ouabain binding. Despite these markers, few proteins from purified transverse tubules can be unequivocally identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this report we have biochemically and immunologically identified rabbit albumin as a major component of purified transverse tubule membranes from rabbit skeletal muscle. Albumin composed between 5.1 and 9.8% (n = 4) of the total protein in purified transverse tubules based on scans of SDS-PAGE. Furthermore, albumin and other serum proteins are present in preparations of transverse tubules and triads but not in light sarcoplasmic reticulum. Extraction of triads with low concentrations of saponin or sodium dodecyl sulfate completely removes albumin without removing intrinsic membrane proteins. Our results suggest that albumin and other serum proteins are present in the lumen of preparations of transverse tubules and albumin may be used as a marker for the transverse tubules when analyzed on SDS gels.

Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins.

A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that 1) its apparent molecular weight is not changed by reduction and alkylation; 2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; 3) binding of lipoproteins is not inhibited by EDTA; and 4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins. It is unlikely that this protein ever binds lipoproteins in vivo; however, its lipoprotein binding activity has facilitated its purification to homogeneity and suggests that this protein has unusual structural features. The role of the 165-kDa protein in Ca2+ homeostasis in the sarcoplasmic reticulum, if any, remains to be determined.

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

[The content of phospholipids and free fatty acids in the sarcoplasmic reticulum membranes in the early stage of an x-ray radiation lesion]. / Soderzhanie fosfolipidov i svobodnykh zhirnykh kislot v membrannakh sarkoplazmaticheskogo retikuluma na rannem étape porazheniia rentgenovskim izlucheniem.

One and 24 h following single X-irradiation (0.21 C/kg) of rabbit hind leg the content of free fatty acids and phospholipid lysoforms increased in the sarcoplasmic reticulum (SR) membrane of skeletal muscles. The results obtained are important in estimating the mechanisms of action of ionizing radiation on the structural and functional properties of SR.

Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle.

We studied the subcellular localization of dystrophin in rabbit skeletal muscle. In Western-blot analysis of membrane preparations, dystrophin was associated with the sarcolemmal fraction, as indicated by cholesterol content and co-purification with ouabain-binding activity and beta-adrenergic receptor. Dystrophin was also found with junctional T-tubules, but not with 'free' T-tubules, longitudinal portions or terminal cisternae of the sarcoplasmic reticulum. Dystrophin was not solubilized by high salt solutions, but it was solubilized by low concentrations of detergents (Triton X-100 and deoxycholate), suggesting that it is a peripheral membrane protein.

Molecular cloning and expression of cDNA encoding the 53,000-dalton glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum.

The 53-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum was purified by lentil lectin affinity chromatography and preparative polyacrylamide gel electrophoresis and partially sequenced. Polyclonal and monoclonal antibodies were raised against the 53-kDa glycoprotein and found to cross-react with the 160-kDa glycoprotein. A combination of antibody and synthetic oligonucleotide screening was used to isolate a cDNA encoding the 53-kDa glycoprotein of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The cDNA encodes a protein of 453 amino acids with Mr of 52,421 and a 19-residue amino-terminal signal sequence. The deduced sequence contains two potential glycosylation sites and is largely hydrophilic. The presence of a glycine-rich sequence in the glycoprotein with homology to mononucleotide binding domains supports earlier observations that the glycoprotein binds ATP with high affinity. Although two sequences appear to be hydrophobic on a hydropathy plot, they are not sufficiently long nor sufficiently hydrophobic to qualify unambiguously as transmembrane sequences. The glycoprotein, like calsequestrin, was shown to be inaccessible to trypsin in intact sarcoplasmic reticulum. It can be eluted from the sarcoplasmic reticulum by extraction with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid under hypotonic conditions. Thus, the glycoprotein appears to be localized entirely in the lumen of the sarcoplasmic reticulum and to be associated with the inner membrane surface through Ca2+-dependent mechanisms. Cotransfection of COS-1 cells with cDNAs encoding the glycoprotein and the Ca2+-ATPase led to expression of both proteins with a common localization in the microsomal fraction. The Ca2+ pumping activity of the microsomes isolated from transfected cells was unaltered by the presence of the glycoprotein. Thus the glycoprotein does not appear to modulate Ca2+-ATPase function.

A silver-reducing component in rat striated muscle. I. Selective localization at the level of the terminal cistern/transverse tubule system. Light and electron microscope studies with a new histochemical procedure.

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (Hg--Ag) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 degrees C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

A silver-reducing component in rat striated muscle. II. Isolated sarcoplasmic reticulum vesicles.

In previous work on rat striated muscle cells a silver-reducing component was found selectively localized at the terminal cistern/transverse tubule system (Tandler and Pellegrino de Iraldi 1989). To further investigate that problem we performed the Hg-Ag argentaffin reaction on a sarcoplasmic reticulum fraction from rat skeletal muscle. Circular profiles corresponding to vesicular structures were found outlined by silver grains. The number of silver "stained" vesicles were less than the total number vesicles stained by conventional procedures. The correlation between argentaffinities in the intact muscle fiber and their subcellular organelles indicated that the Hg-Ag reactive vesicles must be those derived from the terminal cisternae of the sarcoplasmic reticulum. The silver-reducing constituent aggregates in the presence of 1 mM CaCl2 or 0.5 M K cacodylate. The state of aggregation induced by Ca2+ was not affected by incubation with 0.5% Triton X-100 or by 2 mM EDTA, thus suggesting a localization at or near the membrane of the terminal cistern vesicle facing the junctional gap. In Laemmli SDS-acrylamide gels the Hg-Ag reaction stained all proteins in a manner similar to Coomasie blue. It is suggested that the selective histochemical staining is the result of differential reactivities due to steric requirements of the chemical reaction.

Structural characterization of phospholamban in cardiac sarcoplasmic reticulum membranes by cross-linking.

The native form of phospholamban in cardiac sarcoplasmic reticulum membranes was investigated using photosensitive heterobifunctional cross-linkers, both cleavable and noncleavable, and common protein modifiers. The photosensitive heterobifunctional cleavable cross-linker ethyl 4-azidophenyl-1, 4-dithiobutyrimidate was used in native SR vesicles and it cross-linked phospholamban into an apparent phospholamban-phospholamban dimer and into an approximately 110,000-Da species. The phospholamban dimer migrated at approximately 12,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and upon cleavage of the cross-linker before electrophoresis the dimer disappeared. The approximately 110,000-Da cross-linked species was not affected by boiling in sodium dodecyl sulfate prior to electrophoresis. This cross-linked form of phospholamban migrated approximately 5500 Da above the Ca2(+)-ATPase, which was visualized using fluorescein 5'-isothiocynate, a fluorescent marker that binds specifically to the Ca2(+)-ATPase. p-Azidophenacyl bromide, iodoacetic acid, and N-ethylmaleimide, all of which react with sulfhydryl groups, were also employed to further characterize phospholamban in native sarcoplasmic reticulum membranes. Cross-linking with p-azidophenacyl bromide resulted in only monomeric and dimeric forms of phospholamban as observed on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetic acid and N-ethylmalemide were found to be effective in disrupting the pentameric form of phospholamban only when reacted with sodium dodecyl sulfate solubilized sarcoplasmic reticulum. In view of these findings, the amino acid sequence of phospholamban was examined for possible protein-protein interaction sites. Analysis by hydropathic profiling and secondary structure prediction suggests that the region of amino acids 1-14 may form an amphipathic alpha helix and the hydrophobic surface on one of its sites could interact with the reciprocal hydrophobic surface of another protein, such as the Ca2(+)-ATPase.

Complete amino acid sequence of the sarcoplasmic calcium-binding protein (SCP-I) from crayfish (Astacus leptodactylus).

The complete amino acid sequence of the alpha chain of the dimeric sarcoplasmic Ca2+-binding protein (SCP-I = alpha 2) from crayfish (Astacus leptodactylus) has been determined by partial automatic sequencing of the peptides derived from tryptic digests of the protein after citraconylation or treatment with 1,2-cyclohexanedione. Overlapping peptides were obtained by cleavage with o-iodosobenzoic acid, or digestion with Staphylococcus aureus protease, thermolysin and pepsin. The acetylated N-terminus was identified by fast atom bombardment mass spectrometry. The monomeric protein contains 192 amino acids and has an Mr of 21,643. The sequence shows the presence of three calcium-binding sites and perhaps of two others that may be degenerated.

The mechanism of the inotropic action of striatoxin, a novel polypeptide toxin from a marine snail, in isolated cardiac muscle.

1. Striatoxin (StTX), a novel polypeptide from a marine snail, caused a dose-dependent increase in contractility in the isolated atria of guinea-pig and rat in the concentration-range of 2 x 10(-9) to 3 x 10(-8)M and 3 x 10(-6)M, respectively. 2. In guinea-pig atria, the StTX-induced inotropic effect was inhibited by tetrodotoxin but not by cimetidine or chlorpheniramine. Practolol, propranolol or reserpine caused only partial block of this inotropic action. 3. In isolated single cells from rat hearts, StTX caused an increase in the degree and the rate of contraction. 4. In guinea-pig atria, StTX provoked action potentials with a plateau phase of long duration without affecting the maximum rate of rise, the amplitude of action potential and the resting membrane potential. This prolongation was also reversed by tetrodotoxin. 5. In guinea-pig cardiac myocytes, whole-cell patch-clamp experiments showed that StTX slowed Na channel inactivation without affecting the time course of channel activation. The voltage dependence of Na currents was not altered by StTX. 6. The residual currents, but not peak currents were markedly enhanced by StTX. 7. These results suggest that StTX causes prolongation of the action potential duration probably due to slowed inactivation of Na inward currents and enhanced residual currents and that this may result in an increase in Ca2+ availability in cardiac muscle cells. This could explain the cardiotonic action of StTX.

Fast atom bombardment mass spectra of various peptides from sarcoplasmic calcium-binding protein.

The sarcoplasmic calcium-binding protein of the crayfish Astacus leptodactylus is a calciprotein. The primary structure of this protein has been determined. Tryptic digestion of the denaturated protein followed by high-performance liquid chromatographic separation identified essentially eight peptides. The structure of these peptides has been confirmed after amino acid analysis by fast atom bombardment spectra in positive mode.