Triadic proteins of skeletal muscle

Biochemical approaches toward understanding the mechanism of muscle excitation have in recent years been directed to identification and isolation of proteins of the triad junction. The principal protein described—the junctional foot protein (JFP)—was initially identified by morphological criteria and isolated using antibody-affinity chromatography. Subsequently this protein was described as the ryanodine receptor. It has been isolated and incorporated into lipid bilayers as a cation channel. This in its turn has directed attention toward the transverse (T)-tubular junctional constituents. Three approaches employing the JFP as a probe toward identifying these moieties on the T-tubule are described here. The binding of the JFP to the dihydropyridine receptor, which has been hypothesized to be the voltage sensor in excitation-contraction coupling, is also discussed. The detailed architecture and function of T-tubular proteins remain to be resolved.Abbreviations DHP dihydropyridine - GAPD glyceraldehyde 3-phosphate dehydrogenase - IP₃ inositol 1,4,5-trisphosphate - JFP junctional foot protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SR sarcoplasmic reticulum - TC terminal cisterna - T-tubule transverse tubule     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

The structure of Ca(2+) release units in arthropod body muscle indicates an indirect mechanism for excitation-contraction coupling

The relative disposition of ryanodine receptors (RyRs) and L-type Ca(2+) channels was examined in body muscles from three arthropods. In all muscles the disposition of ryanodine receptors in the junctional gap between apposed SR and T tubule elements is highly ordered. By contrast, the junctional membrane of the T tubule is occupied by distinctive large particles that are clustered within the small junctional domain, but show no order in their arrangement. We propose that the large particles of the junctional T tubules represent L-type Ca(2+) channels involved in excitation-contraction (e-c) coupling, based on their similarity in size and location with the L-type Ca(2+) channels or dihydropyridine receptors (DHPRs) of skeletal and cardiac muscle. The random arrangement of DHPRs in arthropod body muscles indicates that there is no close link between them and RyRs. This matches the architecture of vertebrate cardiac muscle and is in keeping with the similarity in e-c coupling mechanisms in cardiac and invertebrate striated muscles.     

Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.     

Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads

Summary Isolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetectedM _r 95 K protein which is distinct from the SR Ca²⁺ pump and phosphorylaseb. TheM _r 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified strong triads (accompanying paper). From these findings, we propose a new model for the triad junction.     

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca²⁺ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca²⁺ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca²⁺ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.     

Transport of the alpha subunit of the voltage gated L‐type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles

Contraction of skeletal muscle is initiated by excitation‐contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca²⁺ release. EC coupling requires L‐type voltage gated Ca₂₊ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule‐independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.      

STUDIES OF THE TRIAD : I. Structure of the Junction in Frog Twitch Fibers

The structure of the junction between sarcoplasmic reticulum (SR) and transverse tubular (T) system at the triad has been studied in twitch fibers of the frog. The junction is formed by flattened surfaces of the SR lateral sacs and the T-system tubule, which face each other at a distance of 120–140 A. At periodic intervals of about 300 A, the SR membrane forms small projections, whose tips are joined to the T system membrane by some amorphous material. The SR projections and the amorphous material are here called SR feet. The feet are disposed in two parallel rows, two such rows being present on either side of the T-system tubule. The junctional area between the feet is apparently empty. The feet cover no more than 30% of the T system surface area and 3% of the total SR area. The functional significance of this interpretation of the junctional structure is discussed.     

The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line

The ryanodine receptor/junctional channel complex (JCC) forms the calcium release channel and foot structures of the sarcoplasmic reticulum. The JCC and the dihydropyridine (DHP) receptor in the transverse tubule are two of the major components involved in excitation-contraction (E-C) coupling in skeletal muscle. The DHP receptor is believed to serve as the voltage sensor in E-C coupling. Both the JCC and DHP receptor, as well as many skeletal muscle-specific contractile protein genes, are expressed in the BC3H1 muscle cell line. In the present study, we find that during differentiation of BC3H1 cells, induced by mitogen withdrawal, induction of the JCC and DHP receptor mRNAs is temporally similar to that of the skeletal muscle contractile protein genes alpha-tropomyosin and alpha-actin. Our data suggest that there is coordinate regulation of both the contractile protein genes (which have been studied in detail previously) and the genes encoding the calcium channels involved in E-C coupling. Induction of both calcium channels is accompanied by profound changes in BC3H1 cell morphology including the development of many components of mature skeletal muscle cells, despite lack of myoblast fusion. Visualized by electron microscopy, the JCC appears as "foot structures" located in the dyad junction between the plasmalemma and the sarcoplasmic reticulum of the BC3H1 cells. Development of foot structures is concomitant with JCC mRNA expression. Expression of the JCC and DHP receptor mRNAs and formation of the foot structures are inhibited specifically by fibroblast growth factor.     

Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.     

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.     

Ultrastructure of the calcium release channel of sarcoplasmic reticulum

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.     

The T-SR junction in contracting single skeletal muscle fibers

The junction between the T system and sarcoplasmic reticulum (SR) of frog skeletal muscle was examined in resting and contracting muscles. Pillars, defined as pairs of electron-opaque lines bounding an electron- lucent interior, were seen spanning the gap between T membrane and SR. Feet, defined previously in images of heavily stained preparations, appear with electron-opaque interiors and as such are distinct from the pillars studied here. Amorphous material was often present in the gap between T membrane and SR. Sometimes the amorphous material appeared as a thin line parallel to the membranes; sometimes it seemed loosely organized at the sites where feet have been reported. Resting single fibers contained 39 +/- 14.3 (mean +/- SD; n = 9 fibers) pillars/micrometer2 of tubule membrane. Single fibers, activated by a potassium-rich solution at 4 degrees C, contained 66 +/- 12.9 pillars/micrometer2 (n = 8) but fibers contracting in response to 2 mM caffeine contained 33 +/- 8.6/micrometer2 (n = 5). Pillar formation occurs when fibers are activated electrically, but not when calcium is released directly from the SR; and so we postulate that pillar formation is a step in excitation-contraction coupling.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle

Calcium release during excitation-contraction coupling of skeletal muscle cells is initiated by the functional interaction of the exterior membrane and the sarcoplasmic reticulum (SR), mediated by the "mechanical" coupling of ryanodine receptors (RyR) and dihydropyridine receptors (DHPR). RyR is the sarcoplasmic reticulum Ca(2+) release channel and DHPR is an L-type calcium channel of exterior membranes (surface membrane and T tubules), which acts as the voltage sensor of excitation-contraction coupling. The two proteins communicate with each other at junctions between SR and exterior membranes called calcium release units and are associated with several proteins of which triadin and calsequestrin are the best characterized. Calcium release units are present in diaphragm muscles and hind limb derived primary cultures of double knock out mice lacking both DHPR and RyR. The junctions show coupling between exterior membranes and SR, and an apparently normal content and disposition of triadin and calsequestrin. Therefore SR-surface docking, targeting of triadin and calsequestrin to the junctional SR domains and the structural organization of the two latter proteins are not affected by lack of DHPR and RyR. Interestingly, simultaneous lack of the two major excitation-contraction coupling proteins results in decrease of calcium release units frequency in the diaphragm, compared with either single knockout mutation.     

Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum

We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85% of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles. Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5% Triton X-100 in the presence of 1 mM CaCl2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 mumol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca2+ release process in excitation-contraction coupling, is now available in the test tube.     

Abnormal features in skeletal muscle from mice lacking mitsugumin29

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.     

A functional identification of cardiac junctional sarcoplasmic reticulum

Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodine-sensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T-) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.     

FREEZE FRACTURE OF SKELETAL MUSCLE FROM THE TARANTULA SPIDER : Structural Differentiations of Sarcoplasmic Reticulum and Transverse Tubular System Membranes

The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.