Neil R. Brandt

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

SummaryIsolated 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 undetectedMr 95 K protein which is distinct from the SR Ca2+ pump and phosphorylaseb. TheMr 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.

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

SummaryThe 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 frament 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.

Ion-induced release of calcium from isolated sarcoplasmic reticulum

SummaryCholine 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−<cacodylate−<isethionate−=methane sulfonate−<methylsulfate−<SCN− for anions, and K+=Na+=Li+<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.

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

Abstract 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.

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

SummaryIt 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.

Identification of two populations of cardiac microsomes with nitrendipine receptors: Correlation of the distribution of dihydropyridine receptors with organelle specific markers☆

Conventional sarcolemma and microsome preparations from rabbit and cat ventricular muscle were fractionated on continuous linear sucrose gradients. The distribution of nitrendipine receptors was compared with the distribution of organelle specific markers. For the conventional sarcolemma preparation, the dihydropyridine receptor distribution matched the pattern for external membrane markers in position and shape. The number of nitrendipine receptors was three times the number of muscarine binding sites (approximately 1.0 pmol/mg protein) at the isopycnic point of the vesicles. In contrast, two populations of vesicles with nitrendipine receptors were found in the microsome preparations. One population banded with the external membrane vesicles at a mean buoyant density of 24% (w/w) sucrose. The specific content of dihydropyridine receptors (0.2 pmol/mg) was 1/5 that for the muscarine receptors. The second and major population followed the distribution of an Mr 300K polypeptide, a marker for the junctional cisternae of the sarcoplasmic reticulum (SR). Muscarine receptors, however, were also present throughout that band, albeit at a reduced specific content (approximately 0.1 pmol/mg) compared to the light vesicles. The nitrendipine specific content increased over threefold from that of the light vesicles such that the relative content (nitrendipine/muscarine) was twice that determined for the conventional sarcolemma preparation. Nitrendipine receptors were not associated with nonjunctional SR or mitochondria. The light and heavy microsome populations were incubated with 0.2 mg digitonin/mg protein, a treatment which preferentially perturbs the isopycnic point of external membrane vesicles. For the light vesicles, the membranes with muscarine and nitrendipine receptors became heavier than the bulk of the SR. In contrast, after digitonin treatment of the heavy vesicle population, the nitrendipine and muscarine receptors and the SR marker appeared to comigrate into a sharpened band at 39% sucrose. The possibility that the dihydropyridine binding sites in the heavy microsome population are on external membrane vesicles physically linked to the junctional SR is discussed.

The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with alpha-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high pH 6.8/pH 8.2 activity ratio (0.4-0.7) and a high level of Ca2+ independent activity (EGTA/Ca2+ = 0.3-0.5). The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a Mr 128,000 polypeptide in the gradients. This polypeptide and a Mr 143,000 polypeptide were labeled with 32P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a Mr 102,000 membrane polypeptide which followed the distribution of Ca2+-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A Mr 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca2+/calmodulin-stimulated manner, including a Mr ca. 72,000 polypeptide found only in the transverse tubule.

Detection and localization of triadin in rat ventricular muscle

SummaryDyads (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 Mr 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 Mr as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 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.

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.