Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium.

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.

Does muscle activation occur by direct mechanical coupling of transverse tubules to sarcoplasmic reticulum?

Our knowledge of the physiological and biochemical constituents of skeletal muscle excitation has increased greatly during the last few years but this has not led to a consensus of the physiological mode of muscle activation. Three hypotheses of transmission, involving either transmitter-receptor interaction or direct mechanical coupling, are still under active consideration. The hypothesis of direct mechanical coupling currently being evaluated proposes that the dihydropyridine receptor in the transverse tubules serves as a voltage sensor that communicates directly with the junctional foot protein/Ca2+ channel of sarcoplasmic reticulum to initiate opening of the channel.

Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum.

The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T-tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization-induced Ca2+ release nor on caffeine-induced Ca2+ release. Since the slow phase of depolarization-induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.

Does phosphorylase kinase control glycogen biosynthesis in skeletal muscle?

Immunoblotting as well as enzyme assays demonstrate the presence of the self-glucosylating protein, glycogenin, in the protein-glycogen complex, in the sarcoplasmic reticulum and in phosphorylase kinase. In all three compartments glycogenin occurs in different, albeit, defined glucosylated forms, which upon deglucosylation are converted into a 42 kDa form. We suggest that phosphorylase kinase might have a dual function in glycogen biogenesis: firstly, control of glycogen degradation in the protein-glycogen complex via phosphorylation of glycogen phosphorylase b; secondly, regulation of glycogen biosynthesis on the sarcoplasmic reticular membranes via phosphorylation and thereby inhibition of glycogen synthase.

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)2--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.

The localization of honey bee thorax trehalase.

Differential and sucrose gradient centrifugation of honey bee thoraces, disrupted by gentle methods and using mannitol-triethanolamine-EDTA buffer at pH 6.5, showed that in the honey bee thorax 92-94.8% of the trehalase was mitochondrial. Since only 92-95% of the cytochrome c oxidase, a known mitochondrial enzyme, was found in the mitochondrial fraction by these methods, it was concluded that honey bee trehalase is totally mitochondrial. Significant amounts of 'microsomal' or 'soluble' trehalase were formed only by harsh methods of thorax disruption and similar 'microsomal' or 'soluble' trehalases were also formed by harsh treatment of purified whole mitochondria. They thus seem to be artifacts of the isolation procedure. Studies (using marker enzymes) with purified intact mitochondria which were dispersed by various chemical, enzymatic, and physical methods showed that the trehalase in the mitochondria was membrane bound and that it was bound to either the outside of the inner membrane or to one of the sides of the outer membrane.

Ion-induced release of calcium from isolated sarcoplasmic reticulum.

Choline Cl addition to either longitudinal reticulum or terminal cisternae of skeletal muscle sarcoplasmic reticulum caused release of Ca2+ which had previously accumulated in the presence of ATP. However the extent of release was considerably greater in terminal cisternae. Ca2+ accumulation and release by terminal cisternae were also observed using chlorotetracycline as a probe for membrane-associated Ca2+. Among a number of salts and ions tested for effectiveness in causing Ca2+ release the order was gluconate- less than cacodylate- less than isethionate- = methane sulfonate- less than methylsulfate- less than SCN- for anions, and K+ = Na+ = Li+ less than choline+ = tetramethylammonium+ for cations. Valinomycin enhanced Ca2+ accumulation in the presence of ATP both in the absence and presence of the releasing agent, choline Cl. The concentration of sucrose in the medium exerted no discernible effect on the rate or extent of Ca2+ release from terminal cisternae. The rate of release was estimated using a stopped-flow mixing apparatus. The rapid phase of release was complete in 6 sec when choline Cl or KSCN were employed to initiate release. Ca2+ efflux was slower when release was initiated by EGTA addition. The estimated rate of release was 4-6 nmol/mg protein/sec. The fluorescent probe, 1-anilino-8-naphthalene sulfonate was employed to estimate the influence of ions on the surface potential of terminal cisternae. A broad inverse correlation was observed between the fluorescence of the probe in the presence of various salts and their ability to induce Ca2+ release.

Intracellular localization of inositol-phospholipid-metabolizing enzymes in rabbit fast-twitch skeletal muscle. Can D-myo-inositol 1,4,5-trisphosphate play a role in excitation-contraction coupling?

Rabbit fast-twitch skeletal muscle microsomes have been separated by isopycnic centrifugation on a linear sucrose gradient into triads and light sarcoplasmic reticulum. In both fractions phosphatidylinositol-kinase activity is found [Varsányi et al. (1986) Biochem. Biophys. Res. Commun. 138, 1395]. In contrast, phosphatidylinositol-4-phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol-4,5-bisphosphate-phosphodiesterase activity shows a biphasic distribution: approximately 50% of the activity is associated with triads and 50% appears in the overlay. Triads have been broken mechanically into transverse tubules and terminal cisternae, then separated by isopycnic sucrose-gradient centrifugation. Both fractions exhibit phosphatidylinositol-kinase activity; the activities of phosphatidylinositol-4-phosphate kinase and phosphatidylinositol-4,5-bisphosphate phosphodiesterase are associated mainly with the transverse tubules. Consequently, in rabbit fast-twitch skeletal muscle all necessary enzymes for production of D-myo-inositol 1,4,5-trisphosphate are associated with transverse tubules. Phosphatidylinositol-4,5-bisphosphate phosphodiesterase associated with triads shows a pH optimum at 6.8. The enzyme is maximally active between pCa 5 and pCa 4. Mg2+ inhibits the enzyme activity half-maximally at about 1 mM. Guanine-nucleotide-binding proteins seem not to be involved in the regulation of enzyme activity; guanosine 5'-[gamma-thio]triphosphate does not influence phosphatidylinositol-4,5-bisphosphate phosphodiesterase activity. It correlates well with the observation that neither alpha 1-adrenergic nor muscarinic receptors have been found in fast-twitch rabbit skeletal muscle. On basis of the respective enzyme activities estimations on maximal phosphatidylinositol turnover were made and a possible involvement of this signal pathway in excitation-contraction coupling has been discussed. Furthermore, calculations show that during a single twitch D-myo-inositol 1,4,5-trisphosphate concentration does not reach more than 2 nM. However, during a 4-s tetanus D-myo-inositol 1,4,5-trisphosphate can accumulate to a level which could effect force generation [Thieleczek and Heilmeyer (1986) Biochem. Biophys. Res. Commun. 135, 662] and aldolase distribution (Thieleczek et al., unpublished results).

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.

Disulfide bonds, N-glycosylation and transmembrane topology of skeletal muscle triadin.

Native triadin is a disulfide linked homopolymer of variable subunit number. Two monoclonal antibodies (mAbs), AE8.91 and GE4.90, recognize cytoplasmic regions of triadin between amino acids 110 and 163 and at the C-terminal 34 amino acids, respectively. Triadin in intact triads is largely unaffected by trypsin, while triads whose membrane has been disrupted by hypotonicity or by treatment with the detergent Triton X-100 yield both soluble and membrane bound fragments. Soluble fragments monitored by mAb GE4.90 appear to be formed sequentially during the course of proteolysis at 28, 16, 10 and 7 kDa in the presence of mercaptoethanol. Higher molecular weight bands are observed under nonreducing conditions. A two-dimensional electrophoresis immunoblot (first nonreducing; second reducing) of the soluble fragments developed with mAb GE4.90 shows the presence of several bands which can be interpreted as containing a dimer formed by a combination of any two of the fragments of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not detect these fragments. This observation indicates that one of the intermolecular disulfide bonds is formed between the identical domains of two triadin molecules at cysteine 671. Immunoblots performed with and without mercaptoethanol of the insoluble fragments using mAb AE8.91 indicate the presence of a dimer formed between identical domains of two triadin intermolecular disulfide linkage at cysteine 270. The glycosidase endo F/N-glycosidase F changed the mobility of intact triadin in TC/triads and its proteolytic fragments detected by mAb GE4.90.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetic parameters of trehalase in whole and disrupted mitochondrial preparations from two insects with asynchronous muscle.

The kinetic parameters of trehalase in honey bee and flesh fly mitochondria were compared. The studies were carried out with whole mitochondria and with mitochondria disrupted in various ways and to different degrees. Honey bee mitochondrial trehalase was significantly activated by Lubrol WX treatment (30.0-fold), by high pH treatment (20.8-fold), and by a treatment consisting of 10 passes through a French press (37.9-fold) but not by the other treatments tried (salt, proteases, Waring blender, and sonication), despite the fact that these treatments also disrupted the mitochondria significantly. The activation effect was on the Vmax. The Km value did not change. Simple breakage of either the outer or inner (or both) membranes was not sufficient to activate trehalase from honey bees, which showed that the activation was not an indirect result of a change in the case with which trehalose can pass through the membranes. Honey bee trehalase is the first trehalase from insects with asynchronous muscle which has been shown to be activatable by physical and chemical methods. Flesh fly mitochondrial trehalase behaved quite differently from the honey bee enzyme in that it could not be activated by any of the techniques tried, even when there were significant amounts of disruption.

Effects of mercaptans upon dihydropyridine binding sites on transverse tubules isolated from triads of rabbit skeletal muscle.

The binding of nitrendipine to transverse (T) tubules isolated from skeletal muscle triads is inhibited by dithiothreitol (KI approximately 0.05 mM) and glutathione (KI approximately 3 mM). The t 1/2's of inhibition (18.3 and 11.5 min, respectively) suggest that these hydrophylic reagents act upon the exposed surface of the vesicles. Dithiothreitol shifts the apparent KD for nitrendipine from 8.5 nM to 30 nM without altering the Bmax extrapolated by Scatchard analysis. That T-tubules isolated by disruption of triad junctions are constrained to have the protoplasmic (P) face uniformly exposed was experimentally confirmed. These studies show that a sulfhydryl residue on the P-face of the T-tubule influences the affinity of the receptor for dihydropyridines.

Dihydropyridine binding sites on transverse tubules isolated from triads of rabbit skeletal muscle.

Receptors for the dihydropyridine class of Ca2+ channel antagonists are present on transverse tubules isolated by mechanical disruption of skeletal muscle triads. These observations account for the previously reported presence of nitrendipine binding sites in heavy sarcoplasmic reticulum. Nitrendipine receptors were not found in the terminal cisternae after disruption of the triad junctions. The number of sites in junctional T-tubules (27 pmol/mg) is only half that reported for transverse tubules isolated as free vesicles (pmol/mg). The presence of nitrendipine receptors in that region of the transverse tubules held in close apposition to the SR cisternae is consistent with the architectural requirements for the Ca2+ induced Ca2+ release phenomenon to be the mechanism of excitation-contraction coupling.

ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle.

A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy.

Inositol polyphosphate-mediated repartitioning of aldolase in skeletal muscle triads and myofibrils.

The effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been hypothesized to be a chemical transmitter in excitation-contraction coupling in skeletal muscle, on aldolase bound to isolated triad junctions were investigated. Fructose-1,6-bisphosphate aldolase was identified as the major specific binding protein for the Ins(1,4,5)P3 analogue glycolaldehyde (2)-1-phospho-D-myo-inositol 4,5-bisphosphate which can form covalent bonds with protein amino groups by reduction of the Schiff's base intermediate with [3H]NaCNBH3. This analogue, Ins(1,4,5) P3, and the inositol polyphosphates inositol 1,3,4,5-tetrakisphosphate and inositol 1,4-bisphosphate were nearly equipotent in selectively releasing membrane bound aldolase with a K0.5 of about 3 microM. The rank order of the K0.5 values was identical to the KI values for inhibition of aldolase. Aldolase was also released by its substrate fructose 1,6-bisphosphate and by 2,3-bisphosphoglycerate. Ins(1,4,5)P3-induced aldolase release did not disrupt the triad junction; glyceraldehyde-3-phosphate dehydrogenase, a known junctional constituent, was displaced only at much higher Ins(1,4,5)P3 concentrations. Ins(1,4,5)P3 was as effective as fructose 1,6-bisphosphate in releasing aldolase from myofibrils. A finite number of binding sites for aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase/mg of triad protein, KD = 23 nM). The junctional foot protein was implicated as an aldolase binding site by affinity chromatography with the junctional foot protein immobilized on Sepharose 4B. The potential consequences of aldolase being bound in the gap between the terminal cisternae and the transverse tubule to inositol polyphosphate and glycolytic metabolism in that local region are discussed.

Determinants of triad junction reformation: identification and isolation of an endogenous promotor for junction reformation in skeletal muscle.

The junction of isolated triads can be mechanically broken by passage through a French press and subsequently reformed by incubation of the isolated organelles with certain salts of weak acids (e.g., K cacodylate, K propionate, and K butyrate). In contrast, other salts (e.g., KCl, K phosphate, and K benzoate) are ineffective in promoting triad formation. An endogenous factor obtained from a muscle homogenate acts in the same manner as these artificial compounds. When rabbit skeletal muscle is homogenized in a KCl solution and centrifuged to remove large cellular components and membrane fractions, an endogenous factor is extracted into the high speed supernatant which promotes the reformation of mechanically broken triads. A three-stage purification of this factor has been achieved using: ammonium sulfate fractionation, adsorption chromatography, and molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunit Mr of 34,000 daltons. This protein has the following characteristics: it exists in 0.1 M KCl as a polymeric substance with an estimated Mr = 123,000 on molecular sieve chromatography and a Mr = 155,000 on sedimentation equilibrium; it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; Both this protein and cacodylate share the property of specifically catalyzing the association and aggregation of junctional proteins which had previously been dissolved by neutral detergent and salt; it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein of Mr = 34K. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominantly a catalytic role in the formation of the triad junction.

Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle.

The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.

Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle.

It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca2(+)-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, "weak," and breakage resistant, "strong," triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.

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

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 undetected Mr 95 K protein which is distinct from the SR Ca2+ pump and phosphorylase b. The Mr 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.