Metabolic Biotinylation as a Probe of Supramolecular Structure of the Triad Junction in Skeletal Muscle

In skeletal muscle excitation–contraction (E–C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle.

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A Molecular Model of Excitation-Contraction Coupling at the Skeletal Muscle Triad Junction via Coassociated Oligomeric Calcium Channels

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1989.tb24096.x ◽

1989 ◽

Vol 560 (1 Calcium Chann) ◽

pp. 185-188 ◽

Cited By ~ 5

Author(s):

LIN HYMEL ◽

HANSGEORG SCHINDLER ◽

MAKOTO INUI ◽

SIDNEY FLEISCHER ◽

JÖRG STREISSNIG ◽

...

Keyword(s):

Skeletal Muscle ◽

Calcium Channels ◽

Molecular Model ◽

Excitation Contraction Coupling ◽

Contraction Coupling ◽

Triad Junction

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Proteins of the Triad Junction of Skeletal Muscle

Transduction in Biological Systems ◽

10.1007/978-1-4684-5736-0_24 ◽

1990 ◽

pp. 361-369

Author(s):

Anthony H. Caswell ◽

Neil R. Brandt ◽

Shu-Rong Wen ◽

Jane A. Talvenheimo

Keyword(s):

Skeletal Muscle ◽

Triad Junction

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Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.127.2.411 ◽

1994 ◽

Vol 127 (2) ◽

pp. 411-423 ◽

Cited By ~ 190

Author(s):

M Radermacher ◽

V Rao ◽

R Grassucci ◽

J Frank ◽

A P Timerman ◽

...

Keyword(s):

Skeletal Muscle ◽

Calcium Release ◽

Structural Integrity ◽

Three Dimensional ◽

Calcium Release Channel ◽

Density Region ◽

Release Channel ◽

Dimensional Reconstruction ◽

Triad Junction ◽

Cytoplasmic Assembly

The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 degrees rotation about an axis in the plane of the specimen grid. The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca(2+)-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.

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The Role of Calpains in Skeletal Muscle Remodeling with Exercise and Inactivity-induced Atrophy

International Journal of Sports Medicine ◽

10.1055/a-1199-7662 ◽

2020 ◽

Vol 41 (14) ◽

pp. 994-1008 ◽

Cited By ~ 1

Author(s):

Hayden W. Hyatt ◽

Scott K. Powers

Keyword(s):

Skeletal Muscle ◽

Cell Signaling ◽

Protein Turnover ◽

Cysteine Proteases ◽

Biologically Active ◽

Membrane Repair ◽

Downstream Signaling ◽

Coupling Protein ◽

Triad Junction ◽

Muscle Remodeling

AbstractCalpains are cysteine proteases expressed in skeletal muscle fibers and other cells. Although calpain was first reported to act as a kinase activating factor in skeletal muscle, the consensus is now that calpains play a canonical role in protein turnover. However, recent evidence reveals new and exciting roles for calpains in skeletal muscle. This review will discuss the functions of calpains in skeletal muscle remodeling in response to both exercise and inactivity-induced muscle atrophy. Calpains participate in protein turnover and muscle remodeling by selectively cleaving target proteins and creating fragmented proteins that can be further degraded by other proteolytic systems. Nonetheless, an often overlooked function of calpains is that calpain-mediated cleavage of proteins can result in fragmented proteins that are biologically active and have the potential to actively influence cell signaling. In this manner, calpains function beyond their roles in protein turnover and influence downstream signaling effects. This review will highlight both the canonical and noncanonical roles that calpains play in skeletal muscle remodeling including sarcomere transformation, membrane repair, triad junction formation, regulation of excitation-contraction coupling, protein turnover, cell signaling, and mitochondrial function. We conclude with a discussion of key unanswered questions regarding the roles that calpains play in skeletal muscle.

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Abnormal Features in Skeletal Muscle from Mice Lacking Mitsugumin29

The Journal of Cell Biology ◽

10.1083/jcb.147.7.1473 ◽

1999 ◽

Vol 147 (7) ◽

pp. 1473-1480 ◽

Cited By ~ 60

Author(s):

Miyuki Nishi ◽

Shinji Komazaki ◽

Nagomi Kurebayashi ◽

Yasuo Ogawa ◽

Tetsuo Noda ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Surface ◽

Membrane Structures ◽

Intracellular Membranes ◽

Transmembrane Segments ◽

Membrane Complex ◽

Junctional Complexes ◽

Triad Junction ◽

Normal Health

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 Ca2+ 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 Ca2+-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.

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Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

Molecular Biology of the Cell ◽

10.1091/mbc.5.10.1105 ◽

1994 ◽

Vol 5 (10) ◽

pp. 1105-1118 ◽

Cited By ~ 56

Author(s):

B E Flucher ◽

S B Andrews ◽

M P Daniels

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Structural Characteristics ◽

Molecular Organization ◽

Muscle Action Potential ◽

T Tubules ◽

Triad Junction ◽

First Contact ◽

The Relationship

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.

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Cryo-Electron Tomography of Isolated Triad Junctions from Skeletal Muscle

Microscopy and Microanalysis ◽

10.1017/s1431927600026544 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 94-95 ◽

Cited By ~ 1

Author(s):

C.-E. Hsieh ◽

M. Marko ◽

B.K. Rath ◽

S. Fleischer ◽

T. Wagenknecht

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Electron Tomography ◽

Mass Density ◽

Tomographic Reconstruction ◽

Three Dimensional ◽

Cryo Electron Tomography ◽

Junctional Complexes ◽

Triad Junction ◽

Triad Junctions

In skeletal muscle, depolarization of the plasma membrane, which is initiated at the neuromuscular junction, is transduced to a rise in cytoplasmic calcium at specialized structures known as triad junctions (TJs). TJs occur in the myofiber’s interior at regions near the z-lines, where transversely oriented tubular invaginations of the plasma membrane (T-tubules) form junctions with two elements of the sarcoplasmic reticulum (SR). Isolation of membrane fractions that are enriched in junctional complexes and which retain function has been reported.Figure 1 shows a region of an electron micrograph containing an isolated TJ in the frozen-hydrated state. in the orientation shown, two SR-derived vesicles sandwich a flattened vesicle derived from the T-tubule. The junctional regions contain a complex distribution of density, presumably due to proteins that are known to be present in TJs. Electron tomography offers the means to determine the three-dimensional mass density from such micrographs, which would greatly aid in their interpretation. Only recently has the automated data collection technology for determining tomograms of non-stained, frozen-hydrated specimens become available. Here we describe the first tomographic reconstruction of a frozen-hydrated triad junction by automated electron tomography.

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Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.93.3.543 ◽

1982 ◽

Vol 93 (3) ◽

pp. 543-550 ◽

Cited By ~ 31

Author(s):

J J Cadwell ◽

A H Caswell

Keyword(s):

Skeletal Muscle ◽

Protein Pair ◽

Specific Marker ◽

Molecular Weights ◽

Single Protein ◽

T Tubules ◽

Triad Junction ◽

Terminal Cisternae ◽

Substantial Degree ◽

Triad Junctions

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38-42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.

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