Location of ryanodine receptor binding site on skeletal muscle triadin.

The binding between intact triadin or expressed triadin peptides and the ryanodine receptor has been investigated using membrane overlay and affinity chromatography. Ryanodine receptor binds to triadin blotted onto nitrocellulose with a KD of 40 nM in a medium containing 150 mM NaCl. The binding is substantially inhibited by hypertonic salt solution. Blot overlay experiments show that ryanodine receptor binds to bacterially expressed peptides, triadin(110-280), triadin(110-267), and triadin(279-674), but to no other moieties of the protein (numbers in parentheses are the residue positions). This binding is strongly inhibited by hypertonic salt solution. The same three triadin peptides as well as triadin(68-267), when attached to a glutathione column, bind to the ryanodine receptor. However, triadin(110-280) binds with high affinity, while triadin(68-267), triadin(110-267), and triadin(279-674) bind with low affinity. Triadin(258-280), triadin(267-280), and triadin(258-299) all bind to the ryanodine receptor with high affinity. On the other hand, a construct containing triadin(267-280), but preceded by nine residues of heterologous amino acids, does not bind significantly. These observations indicate two types of binding between triadin and the ryanodine receptor: (1) a low-affinity ionic interaction of large portions of triadin; (2) a specific high-affinity binding of a short relatively hydrophobic segment. The binding of this segment is probably the physiologically important domain for attachment between triadin and the ryanodine receptor.

Binding of the catalytic subunit of protein phosphatase-1 to the ryanodine-sensitive calcium release channel protein.

A number of studies have reported that the activity of the ryanodine-sensitive calcium release channel (ryanodine receptor) in the junctional sarcoplasmic reticulum of skeletal and cardiac muscle can be modulated by protein phosphorylation-dephosphorylation through activation of endogenous protein kinases and/or by addition of exogenous protein kinases and protein phosphatases. In this study, we have investigated the possibility that protein phosphatase-1 (PP1) is targeted to the junctional sarcoplasmic reticulum by the direct isolation of PP1-binding proteins on PP1-Sepharose affinity columns. The results show that the ryanodine receptor of both skeletal and cardiac muscle bind to this affinity support, and are released at supraphysiological salt concentrations in a relatively pure state. Reciprocal experiments demonstrated that PP1 binds to the immobilized muscle ryanodine receptor. The direct binding of PP1 to the ryanodine receptor was supported by the finding that tryptic fragments of the receptor were retained on PP1-Sepharose. The ability of PP1 to dephosphorylate the ryanodine receptor that was phosphorylated by protein kinase A was also demonstrated. These studies show that PP1 is targeted to the junctional sarcoplasmic reticulum by binding to the ryanodine receptor, and provide a biochemical basis for the possibility that PP1 may play a role in the regulation of calcium flux via protein phosphorylation-dephosphorylation mechanisms.

Hypertrophy decreases cardiac KATP channel responsiveness to exogenous and locally generated (glycolytic) ATP.

This study tests the hypothesis that glycolytic regulation of KATP channel activity is altered in myocardial hypertrophy. Left ventricular (LV) subendocardial myocytes were isolated from cats with normal or left ventricular hypertrophied hearts (LVH). Saponin-permeabilized open cell-attached patch configurations of normal and LVH cells were exposed to an exogenous ATP consuming system containing hexokinase and 2-deoxyglucose. Phosphoenol pyruvate (PEP, substrate for the last ATP producing step in glycolysis) was applied extracellularly; ADP was present. In both cell types, KATP channels were activated in the absence of PEP, inhibited when PEP was added and activated again when PEP was removed, indicating the cells retained metabolic integrity and generated ATP in the proximity of their KATP channels. Single channel conductance in the absence of PEP was similar (70 pS, normal; 66 pS, LVH). However, LVH KATP channels showed enhanced activity (P0=0.50+/-0.03); normal (0.41+/-0.03) in PEP absence (P<0. 05). PEP responsiveness was reduced in LVH, with IC50, PEP increased to 23 microM; (11 microM normal). Lactate failed to activate KATP channels in both cell types. The concentration-P0 response curves obtained during exposure of open cells to exogenous ATP also revealed reduced responsiveness to ATP of LVH KATP channels (IC50, ATP=283 microM LVH; 93 microM normal). Our data indicate myocardial hypertrophy increases the maximal activity of KATP channels in the absence of ATP and reduces their responsiveness to ATP, including locally generated glycolytic ATP. These alterations in metabolic regulation of myocardial electrophysiology may contribute to diversity of action potential shortening in hypertrophied hearts during acute ischemia.

Binding sites of monoclonal antibodies and dihydropyridine receptor alpha 1 subunit cytoplasmic II-III loop on skeletal muscle triadin fusion peptides.

Triadin binds to the dihydropyridine receptor (DHPr) and the junction foot protein (JFP) in Western blot protein overlay experiments. Fusion peptides were synthesized using an expression system, pGSTag, which includes a protein kinase A phosphorylation site. Expressed peptides are DHPr664-799 encoding rabbit skeletal DHPr alpha1 subunit amino acids 664-799, triadin 1 (1-49), triadin 2 (68-389), triadin 2' (110-389), triadin 2a (68-278), triadin 2a1 (67-163), triadin 2a2 (165-240), triadin 2b (242-389), triadin 2b1 (242-299), triadin 3 (370-706), triadin 3a (370-562), triadin 3b (551-706), triadin 3b1 (551-672), and triadin 3b2 (673-706) (the numbers in parentheses correspond to the amino acid sequence of triadin). The triadin monoclonal antibodies, GE4.90 and AE8.91, bind to intact triadic vesicles as well as to vesicle fragments prepared after treatment with Triton X-100, indicating that they have cytoplasmic epitopes. MAb AE8.91 binds to triadin 2, 2', 2a, and 2a1, while mAb GE4.90 binds to triadin 3, 3b, and 3b2 indicating that residues 110-163 and the C-terminal 34 amino acids contain cytoplasmic domains. Radiolabeled DHPr664-799 binds to triadin in intact vesicles under nonreducing and reducing conditions. It binds to triadin fusion peptides, triadin 2, 2a, 3, 3b, and 3b1, but no to triadin 1 or triadin 3b2. The binding to triadin 2a is the most prominent. Direct binding between DHPr-644-799 and JFP was not seen. These experimental findings indicate that triadin contains an extensive cytoplasmic domain that binds to the domain of DHPr which is considered critical for signal transmission during skeletal muscle excitation-contraction sampling.

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)

Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats.

The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.

Ryanodine receptor-ankyrin interaction regulates internal Ca2+ release in mouse T-lymphoma cells.

In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Analysis of the binding of [3H]ryanodine to these internal vesicles reveals the presence of a single, low affinity binding site with a dissociation constant (Kd) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of [3H]ryanodine to its vesicle receptor at least 5-fold (Kd approximately 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca2+ release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca2+ release. Immunoblot analyses using a monoclonal mouse antiryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Double immunofluorescence staining and laser confocal microscopic analysis show that the ryanodine receptor is preferentially accumulated underneath surface receptor-capped structures. T-lymphoma ryanodine receptor was isolated (with an apparent sedimentation coefficient of 30 S) by extraction of the light density vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradient centrifugation. Further analysis indicates that specific, high affinity binding occurs between ankyrin and this 30 S lymphoma ryanodine receptor (Kd = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca2+ release during lymphocyte activation.

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 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.

Extraction of junctional complexes from triad junctions of rabbit skeletal muscle.

Triadin in skeletal muscle exists as a disulfide linked oligomer. It does not dissolve well in CHAPS detergent even in 1 M KCl, but is solubilized after reduction to its monomer by the addition of 2-mercaptoethanol. Purified reduced triadin is not retained on a hydroxylapatite column in the presence of 30 mM Potassium phosphate, while the junctional foot protein and dihydropyridine receptor purified in the absence of triadin are both retained. In contrast, triadin solubilized as a detergent extract of reduced triadic vesicles is retained by the hydroxylapatite column and elutes concomitantly with the junctional foot protein and dihydropyridine receptor. These findings contrast with the observation that native non-reduced triadin is tightly bound to hydroxylapatite and can be separated from the dihydropyridine receptor and the junctional foot protein with elevated potassium phosphate concentrations. Triadin derived from a detergent extract of reduced vesicles is retained with the hydroxytapatite column in the presence of 180 mM potassium phosphate (0 KCl) which eluted a portion of the junctional foot protein and dihydropyridine receptor. Triadin can then be eluted with the remaining portion of junctional foot protein and dihydropyridine receptor upon the addition of KCl (820 mM) to the 180 mM potassium phosphate medium. Gel electrophoresis confirmed the enrichment of junctional proteins in the 180 mM KPi/820 mM KCl eluate. Rate zonal centrifugation of the 180 mM KPi/820 mM KCl eluate shows that a portion of triadin co-migrates with the dihydropyridine receptor indicative of a much higher molecular weight entity than monomeric triadin. Triadin and the dihydropyridine receptor were, however, separated from the junctional foot protein on rate zonal centrifugation. The dissociated proteins of the complex elute from hydroxylapatite columns similar to the purified proteins. Triadin in the high salt hydroxylapatite extract could also be immunoprecipitated by a monoclonal antibody to the junctional foot protein. Furthermore, the dihydropyridine receptor is immunoprecipitated by a monoclonal antibody directed against triadin providing another indication of a complex between the three proteins. Collectively, these results demonstrate a role for triadin as the linkage between the junctional foot protein and dihydropyridine receptor creating a ternary complex at the triad junction in skeletal muscle.

The involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vesicles in mouse T-lymphoma cells.

Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.

Detection and localization of triadin in rat ventricular muscle.

Dyads (transverse tubule--junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M(r) 102K Ca2+ ATPase were associated with a diffuse protein band (22-30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M(r) as triadin. Western blots of muscle microsomes from preparations which had been treated with 100 mM iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of M(r) 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction.

The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.

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.

Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles.

A monoclonal antibody, GE 4.90, has been produced following immunization of mice with the 95-kDa protein (triadin) of terminal cisternae of rabbit fast skeletal muscle isolated in nondenaturing detergent. The antibody binds to a protein of Mr95K in Western blots of microsomal vesicles electrophoresed in the presence of mercaptoethanol. The greatest intensity of the immunoblot reaction is to enriched terminal cisternae vesicles while little binding is seen to longitudinal reticulum and transverse tubules. The content of antigen in different microsomal subfractions has been estimated by immunoassay: terminal cisternae/triads contain 5.6 micrograms/mg of protein while heavy terminal cisternae contain 32 micrograms/mg. The molar content of triadin in vesicles is approximately the same as that of the ryanodine receptor. When Western blots of gels of terminal cisternae are run in nonreducing conditions, little protein of Mr95K is visible. A number of bands, however, forming a ladder of higher molecular weight are discerned, indicating that the 95-kDa protein forms a disulfide-linked homopolymer. A biotinylated aromatic disulfide reagent (biotin-HPDP) labels the 95-kDa protein, the junctional foot protein, and the Mr 106K protein described by others as a Ca(2+)-release channel (SG 106). This latter protein migrates in gel electrophoresis under nonreducing conditions at a molecular weight different from that of the 95-kDa protein. We did not detect any alteration of binding of the 95-kDa protein to the dihydropyridine receptor or junctional foot protein dependent on the state of oxidation of cysteine residues of either triadin or receptor protein used as the overlay probe.

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.