Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.     

Primary structure of the target of calcium vector protein of amphioxus

CaVPT, a target protein of Ca2(+)-vector from amphioxus muscle, was purified from its complex with CaVP after dissociation by 6 M urea and chromatographies on DEAE-cellulose and calmodulin-Sepharose. The amino acid sequence of CaVPT has been determined. The protein is composed of 243 residues and possesses an unblocked N terminus. Its molecular weight is 26,621, distinctly lower than the apparent molecular weight deduced from electrophoresis on sodium dodecyl sulfate-containing gels. CaVPT contains a potential Asn-linked glycosylation site, four potential protein kinase C phosphorylation sites, and two casein kinase II phosphorylation sites. From the sequence the following three particular domains can be inferred: a collagen-like N-terminal segment, rich in Pro and Ala, that resembles the N-terminal segment of skeletal muscle myosin light chain kinase; next to it (from residues 33 to 50) is located a strongly amphiphilic and basic alpha-helical segment which likely binds the calcium vector protein since a proteolytic cut after Arg50, occurring occasionally during the purification of CaVPT, impairs the binding to immobilized calmodulin. This segment is followed by two immunoglobulin folds. The two immunoglobulin folds typically belong to the C2 subclass and particularly resemble those present in the neural cell surface adhesion molecules NCAM, L1, F11, MAG, TAG-1, fasciclin II, and amalgam. Recently, the presence of immunoglobulin folds of this type has been reported in some intracellular muscular proteins, namely in smooth muscle myosin light chain kinase, striated muscle C protein and titin, as well as in the nematode 600-kDa protein twitchin. From this structural study we can formulate the working hypothesis that CaVPT acts on the structure of the thick filament in muscle or regulates, perhaps via other immunoglobulin fold-containing proteins.     

Isolation and characterization of a new Mr 18,000 protein with calcium vector properties in amphioxus muscle and identification of its endogenous target protein

A new Ca2+-binding protein, called CaVP, has been detected in muscle of the cephalochordate amphioxus and purified to electrophoretic homogeneity. The Mr 18,000 protein (pI = 4.9) binds 2 Ca2+ atoms in a noncooperative way with an intrinsic binding constant of 8.2 X 10(6) M-1. Ca2+, but not Mg2+, induces a 10% increase in alpha-helical content in the metal-free protein. CaVP does not interact with chlorpromazine, but forms a Ca2+-dependent complex with melittin. In situ, CaVP forms a high affinity Ca2+-dependent complex with an Mr 36,000 protein present in muscle extracts of amphioxus. This complex has been purified by gel filtration and ion exchange chromatography, and the target protein further purified after dissociation of the complex in the presence of Ca2+-chelating agents and 6 M urea. The nearly pure Mr 36,000 protein also forms a Ca2+-dependent complex with calmodulin which, however, is less stable during electrophoresis than the CaVP-Mr 36,000 protein complex. Amphioxus CaVP does not substitute for calmodulin in a specific enzyme assay nor for troponin C in restoring Ca2+ sensitivity to skinned muscle fibers. Its polyclonal antibody does not cross-react with the latter two activators. No immunological cross-reacting counterpart of CaVP was found in organs of fish and rat. Its relative abundance in amphioxus muscle indicates that CaVP must underlie an important new limb of Ca2+ regulation in this particular muscle.     

Comparative molecular modeling of Amphioxus calcium vector protein with calmodulin and troponin C

Calcium vector protein (CaVP), a new protein isolated from Amphioxus muscle, binds in a Ca2(+)-regulated manner to a 27 kd target protein, named CaVPT, whose function has not been elucidated yet. CaVP bears significant sequence homology to both calmodulin and skeletal muscle troponin C, especially in the C-terminal half of the molecule, which presumably contains the two functional Ca2(+)-binding sites. The N-terminal half contains two abortive EF-hands and is intramolecularly crosslinked with a disulfide bond. Using the crystallographic structures of calmodulin and striated muscle troponin C as a framework, we constructed two different three-dimensional models of CaVP and modeled the intramolecular disulfide bridge. The modeling based upon the coordinates of calmodulin yields a Ca2(+)-filled sites configuration in the N-terminal half of the molecule, even though no Ca2+ is bound in this half, whereas the troponin C-derived model generates a Ca2(+)-empty sites configuration. The models predict that neither is the Ca2(+)-filled nor in the Ca2(+)-empty sites conformation is there any steric and/or energetic obstacle for the formation of the disulfide bridge and that the disulfide bond is poorly accessible to reducing reagents. The optical properties of the Trp and Tyr residues of CaVP indicate that the calmodulin-derived model represents the most plausible prediction.     

Gut CaVP is an innate immune protein against bacterial challenge in amphioxus Branchiostoma belcheri

The importance of calcium-binding proteins in immune response of vertebrates is determined, but whether they have the role in invertebrates is largely unknown. In the present study, phylogenetic analysis indicated that calcium vector protein (CaVP), a protein unique to amphioxus, shared 68% similarity in amino acid sequence with human and mouse calmodulin (CaM). CaVP cDNA was cloned into a bacterial vector pET-32a, and its His-tagged fusion protein was produced in Eschherichia coli cells (BL21). The recombinant CaVP was purified by Ni-NTA column and SDS-PAGE, and then utilized for antibody preparing. The prepared antibodies could recognize amphioxus CaVP with high specificity. Further analysis by Western blotting showed that CaVP was detected in muscle and humoral fluid of normal animals and appeared in gut of bacterial immunized or challenged amphioxus. Interestingly, gut CaVP was significantly higher in a healthy sub-group than a wounded sub-group post bacterial challenge. This response was detected strongly in immunization and challenge by the same Gram-negative bacterium Vibro parahaemolyticus and weakly in immunization by V.?parahaemolyticus and then challenge by Gram-negative Aeromonas hydrophila, whereas no any feedback was found in immunization by V.?parahaemolyticus and challenge by Gram-positive Staphylococcus aureus. These findings indicate the importance of gut CaVP in response to bacterial challenge.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Sarcoplasmic calcium-binding proteins in protochordate and cyclostome muscle

Summary A sarcoplasmic calcium-binding protein (SCP) has been purified from the muscle of the protochordate Amphioxus and shown to be more similar to invertebrate SCP's than to their counterpart found in vertebrates, i.e. parvalbumins. The Amphioxus protein has a pI of 4.9, is rich in tyrosine and tryptophan, has a molecular weight of 22,000 and binds strongly 2Ca²⁺ with a pK of 7.88. Magnesium competes with calcium for only one of the two metal-binding sites and induces positive cooperativity in Ca²⁺ binding.In cyclostome muscle (lamprey and hagfish), no protein with high affinity for Ca²⁺ or Mg²⁺ could be found, irrespective of molecular weight. Instead, a protein with moderate affinity for Ca²⁺ (10⁵ m ^–1) was detected: it has a molecular weight of 60,000 and might be quite ubiquitous, as the presence of a similar protein has been reported both in red and white muscle of vertebrates such as chicken and rabbit.     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca²⁺. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca²⁺ is linked.     

Genomic structure of the amphioxus calcium vector protein.

Calcium vector protein (CaVP) is an EF-hand Ca(2+)-binding protein, which is unique to the protochordate, amphioxus. CaVP is supposed to act as a Ca(2+) signal transductor, but its exact function remains unknown. Not only its function but also its exact evolutionary relationship to other Ca(2+)-binding proteins is unclear. To investigate the evolution of CaVP, we have determined the complete sequences of CaVP cDNAs from two amphioxus species, Branchiostoma lanceolatum and B. floridae, whose open reading frame cDNA and amino acid sequences show 96.5 and 98.2% identity, respectively. We have also elucidated the structure of the gene of B. floridae CaVP, which is made up of seven exons and six introns. The positions of four of the six introns (introns 1, 2, 3, and 5) are identical with those of calmodulin, troponin C, and the Spec protein of the sea urchin. These latter proteins belong to the so-called troponin C superfamily (TnC superfamily) and thus CaVP likely also belongs to this family. Intron 6 is positioned in the 3' noncoding region and is unique to CaVP, so it may represent a landmark of the CaVP lineage only. The position of intron 4 is not conserved in the genes of the TnC superfamily or CaVP, and seems to result from either intron sliding or the addition of an intron (randomly inserted into or close to domain III) to the genes of the TnC superfamily during their evolution.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Ca channels and skeletal muscle diseases

When observed under a microscope, skeletal muscle exhibits striations due to the highly organized arrangement of muscle proteins that interact with one another to induce muscle contraction. Muscle contraction requires transient increases in intracellular ‘Ca²⁺’ concentration. In this review, Ca²⁺ channels contributing to the functional integrity of intracellular Ca²⁺-release and extracellular Ca²⁺-entry during skeletal muscle contraction are reviewed in terms of their properties, newly emerging ancillary proteins to them, and their abnormalities related to human skeletal muscle diseases. Finally, the aim of this review is to show the big picture of the correlation among Ca²⁺ channels that participate in the Ca²⁺ homeostasis in skeletal muscle.     

Electrophoretic and functional identification of two troponin C isoforms in toad skeletal muscle fibers

The differential sensitivity of frog twitch and slow-tonic fibers to Ca²⁺ and Sr²⁺ suggests that these two fiber types express different troponin C (TnC) isoforms. To date, only one TnC isoform from anurans (resembling the mammalian fast-twitch isoform) has been isolated and characterized. In this study, we examined the possibility that anuran striated muscle contains more than one TnC isoform. Toward this end, we determined the TnC isoform composition of 198 single fibers from the rectus abdominis of the cane toad (a mixed slow-tonic and twitch muscle) and of toad cardiac muscle using a method that enables the identification of TnC isoforms on the basis of the effect of Ca²⁺ on their electrophoretic mobility. The fibers were typed according to their myosin heavy chain (MHC) isoform composition. The data indicate that striated muscle of the cane toad contains two TnC isoforms, one of which (TnC-t) is present in all fibers displaying only twitch MHC isoforms and the other of which (TnC-T/c) is present in fibers displaying the tonic MHC isoform and in cardiac muscle. For a subpopulation of 15 fibers, the TnC isoform composition was also compared with Ca²⁺ and Sr²⁺ activation characteristics. Fibers containing the TnC-T/c isoform were 3-fold more sensitive to Ca²⁺, 40-fold more sensitive to Sr²⁺, and responded to a 4.6-fold broader range of [Ca²⁺] than did fibers containing the TnC-t isoform. The Ca²⁺ activation properties of toad fibers containing the TnC-T/c isoform appear to be consistent with the previously reported physiological characteristics of amphibian slow-tonic muscle fibers. myofibrillar proteins; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; alanine SDS-PAGE; hybrid fibers; Ca²⁺-binding proteins; single fiber; muscle protein polymorphism; fiber type     

The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I)

1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca²⁺ ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd²⁺ was almost as effective as Ca²⁺ in producing the spectral changes. Other bivalent metal ions, particularly Mg²⁺, were less effective. 4. Binding of Ca²⁺ by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca²⁺-saturated form of the protein was more retarded on gel filtration than the Ca²⁺-free form. 5. In the presence of Ca²⁺ the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0–8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

Ca2+ attenuates nucleation activity of leiomodin

A transient increase in Ca²⁺ concentration in sarcomeres is essential for their proper function. Ca²⁺ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca²⁺ sensitive. However, the effects of Ca²⁺ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca²⁺. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca²⁺. We propose that Ca²⁺ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca²⁺ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa²⁺ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca²⁺ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca²⁺ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca²⁺ in the regulation of the sarcomeric protein interactions.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits

A Ca²⁺-activated proteolytic enzyme ¹ that partially degrades myofibrials was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca²⁺-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibris prepared from muscles vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and α-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca²⁺-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occuring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additionally the electrophoretic banding pattern of Ca²⁺-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca²⁺-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in membrane organization occur as myoblast fusion allows the formation of multinucleated muscle fibers. In addition to cell fusion events, differentiation of myotubes involves establishment of specialized membrane structures (1, 2). The transverse tubular invagination of sarcolemmal membrane and the intracellular membrane network known as the sarcoplasmic reticulum are two highly organized membrane architectures in cardiac and skeletal muscle. Establishment of these intricate membrane compartments requires extensive remodeling of the immature myoblast membranes. Dynamic membrane remodeling also contributes to many physiologic processes in mature muscle, including Ca²⁺ signaling, trafficking of glucose transporter (GLUT4), and other membrane internalization events involving caveolae structures (3-6). Although defects in membrane integrity have been linked to various forms of muscular dystrophy (7, 8), the molecular machinery regulating these specific membrane recycling and remodeling events in striated muscle is not well defined.The large tripartite motif (TRIM)⁵ family of proteins is involved in numerous cellular functions in a wide variety of cell types. Members of this protein family contain signature motifs that include a RING finger, a zinc binding moiety (B-box), and a coiled coil structure (RBCC), which invariably comprise the amino-terminal domain of TRIM family members (9). The carboxyl-terminal sequence of TRIM proteins is variable; in some cases a subfamily of TRIM proteins contains a SPRY domain, a sequence first observed in the ryanodine receptor Ca²⁺ channel in the sarcoplasmic reticulum membrane of excitable cells (10). Extensive studies have revealed that protein-protein interactions in the cytosol mediate the defined functions of TRIM proteins. For example, the ubiquitin E3 ligase enzymatic activity of several TRIM family members requires the B-box motif (11, 12). Recent studies have also indicated a role for TRIM proteins in defense against events involving membrane penetration, such as protection against infection by various viruses, including human immunodeficiency virus (13-15). Although most of the studies concentrate on the cytosolic action of TRIM, limited reports have investigated the role of TRIM proteins in membrane signaling or recycling.We have previously established an immunoproteomics approach that allows definition of novel components involved in myogenesis, Ca²⁺ signaling, and maintenance of membrane integrity in striated muscle (16). Using this approach, we have shown that junctophilin is a structural protein that establishes functional communication between sarcoplasmic reticulum and transverse tubule membranes at triad and dyad junctions in striated muscle (17-19). Further studies identified mitsugumin 29, a synaptophysin-related protein that is essential for biogenesis of triad membrane structures and Ca²⁺ signaling in skeletal muscle (20, 21). Screening of this immunoproteomics library led to the recent identification of MG53, a muscle-specific TRIM family protein (22). Domain homology analysis revealed that MG53 contains the prototypical RBCC motifs plus a SPRY domain at the carboxyl terminus. Genetic knock-out and functional studies reveal that MG53 nucleates the assembly of the sarcolemmal membrane repair machinery to restore cellular integrity following acute damage to the muscle fiber (22).Here we present evidence illustrating that MG53, in contrast to other known TRIM proteins, can localize to intracellular vesicles and the sarcolemmal membrane. A functional interaction between MG53 and caveolin-3, another muscle-specific protein, plays an essential role in regulating the dynamic process of membrane budding and exocytosis in skeletal muscle.     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

Expression of genes encoding Ca exporting proteins in freshwater crayfish Procambarus clarkii during cold exposure

1.

This study examined expression of two primary transmembrane Ca²⁺ export proteins (plasma membrane Ca²⁺ ATPase, (PMCA); Na⁺/Ca²⁺ exchanger, sodium/calcium exchanger (NCX)) in epithelial (antennal gland, kidney) and non-epithelial (axial abdominal muscle) tissues of the freshwater crayfish Procambarus clarkii following exposure (28 days) to 4 °C (compared with control 23 °C).