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Nicole A. Beard - One of the best experts on this subject based on the ideXlab platform.

  • Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling ARTICLE
    2013
    Co-Authors: Sanjeewa Goonasekera, Nicole A. Beard, Angela F. Dulhunty, Linda Groom, Isabelle Marty, Takashi Kimura, Alla Lyfenko, Andrew Rosenfeld, Robert T. Dirksen
    Abstract:

    Ca 2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca 2+ to the release machinery. However, the potential impact of the Triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that Triadin binding to RyR1 is critically important for rapid Ca 2+ release during excitation–contraction coupling. To assess the functional impact of the Triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that Triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/ E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca 2+ release were reduced in proportion to the degree of interruption in Triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of Triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin an

  • Triadin peptide does not activate mutant or native RyR1, or when its RyR1 binding site is blocked.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A–B) 3 s traces of RyR1 channel activity at −40 mV. Channels are opening downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). (A) RyR1 ΔM1,2,3 mutants (with Trisk 95 binding residues mutated) in absence of Triadin peptide and in the presence of trans 63 nM and 252 nM peptide. (B) Native RyR1 in the absence and presence of trans 63 nM Triadin peptide. Average open probability (Po) of data, collected at −40 mV and +40 mV (n = 6–10) is displayed below each trace. (C) [3H]ryanodine binding to purified RyR with Triadin binding blocking peptide (RyR14871–4910 ) in the absence and presence of 63 nM Triadin peptide. [3H]ryanodine binding is measured as pmol ryanodine/mg RyR1 and data is expressed relative to binding recorded in the absence of either peptide (rel ryanodine binding). All values are presented as relative to control. Asterisks (*) indicate a significant difference (p≤0.05) in binding from RyR1 control (no peptide); crosshatch (#) indicates a significant difference (p≤0.05) in binding from RyR1 in the presence of RyR14871–4910.

  • Channel open probability parameter values for native and purified RyR1 measured in the presence and absence of 63 nM Triadin peptide and 63 nM full length Trisk 95.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    The average open probability (Po) values calculated from lipid bilayer experiments are shown. Po for each channel type and presence or absence of peptide/Trisk 95 is measured from 90 s of activity at both +40 mV and −40 mV (pooled). The fold change is the Po value after trans addition of Triadin peptide or full length Trisk 95, relative to Po in the absence of Triadin peptide/Trisk 95. Trisk 95 was isolated from rabbit skeletal muscle and these data (in italics) have been previously published in [13] and are included here for comparison. Significant (p≤0.05) differences in Po upon addition of Triadin peptide or Trisk 95 are indicated for each condition compared to its control (*).

  • Triadin peptide modulates purified RyR1 single channel open time, closed time and closed frequency.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A) Average data for open probability (Po) in presence of Triadin200–232 and Triadin200–231 (n = 10), data from both peptides combined (see results text, collectively termed Triadin peptide, n = 11–20) for each of the following parameters; open time (To), close time (Tc) and open frequency (Fo), collected at −40 mV and +40 mV. All data is expressed as relative mean data (Log rel (parameter)). Relative mean Po (log rel Po) is the average of differences between the log10 of the Po in the presence of either 63 nM or 252 nM Triadin peptide (log10 PoPep) and log10 of the control Po (log10 PoCon) for each channel. Log rel To is log10 ToPep-log10ToCon, log rel Tc is log10 TcPep-log10 TcCon and log rel Fo is log10 FoPep-log10 FoCon. (B) [3H]ryanodine binding to purified RyR1 in the absence and presence of 63 nM of Triadin peptide. [3H]ryanodine binding is measured as pmol ryanodine/mg RyR1. Data is expressed relative to binding recorded in the absence of peptides (rel ryanodine binding). Significance (p≤0.05) is indicated for each concentration compared to activity recorded prior to addition of peptide (*).

  • Triadin peptides modulate purified RyR1.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A) Running histogram of a typical bilayer experiment. Open probability (Po) was measured every 30 s throughout the lifetime of the experiment before and after the addition of 63 nM and then 252 nM Triadin200–231 peptide (arrows) at +40 mV (light grey bins) and −40 mV (dark grey bins). Data averages for each condition are shown as horizontal broken lines for +40 mV and −40 mV, and median is presented as a horizontal solid line. (B, C) 3 s traces of purified RyR1 channel activity. Activity was recorded at +40 mV (left) where channels are opening upward from zero current (c, continuous line) to maximum open conductance (o, broken line) and at −40 mV (right), where channel openings are downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). Top panel – control recording of purified RyR1 prior to the addition of Triadin peptide; middle and bottom panel – after the addition of 63 nM and 252 nM Triadin peptide to the trans chamber. (B) Shows addition of Triadin200–232 peptide, and (C) shows addition of Triadin200–231. Control Po in the absence of peptide was 0.46±0.12 at +40 mV and 0.38±0.17 at −40 mV (for Triadin200–232), and 0.14±0.07 at +40 mV and 0.29±0.17 at −40 mV (for Triadin200–231).

Angela F. Dulhunty - One of the best experts on this subject based on the ideXlab platform.

  • Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling ARTICLE
    2013
    Co-Authors: Sanjeewa Goonasekera, Nicole A. Beard, Angela F. Dulhunty, Linda Groom, Isabelle Marty, Takashi Kimura, Alla Lyfenko, Andrew Rosenfeld, Robert T. Dirksen
    Abstract:

    Ca 2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca 2+ to the release machinery. However, the potential impact of the Triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that Triadin binding to RyR1 is critically important for rapid Ca 2+ release during excitation–contraction coupling. To assess the functional impact of the Triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that Triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/ E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca 2+ release were reduced in proportion to the degree of interruption in Triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of Triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin an

  • Triadin peptide does not activate mutant or native RyR1, or when its RyR1 binding site is blocked.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A–B) 3 s traces of RyR1 channel activity at −40 mV. Channels are opening downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). (A) RyR1 ΔM1,2,3 mutants (with Trisk 95 binding residues mutated) in absence of Triadin peptide and in the presence of trans 63 nM and 252 nM peptide. (B) Native RyR1 in the absence and presence of trans 63 nM Triadin peptide. Average open probability (Po) of data, collected at −40 mV and +40 mV (n = 6–10) is displayed below each trace. (C) [3H]ryanodine binding to purified RyR with Triadin binding blocking peptide (RyR14871–4910 ) in the absence and presence of 63 nM Triadin peptide. [3H]ryanodine binding is measured as pmol ryanodine/mg RyR1 and data is expressed relative to binding recorded in the absence of either peptide (rel ryanodine binding). All values are presented as relative to control. Asterisks (*) indicate a significant difference (p≤0.05) in binding from RyR1 control (no peptide); crosshatch (#) indicates a significant difference (p≤0.05) in binding from RyR1 in the presence of RyR14871–4910.

  • Channel open probability parameter values for native and purified RyR1 measured in the presence and absence of 63 nM Triadin peptide and 63 nM full length Trisk 95.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    The average open probability (Po) values calculated from lipid bilayer experiments are shown. Po for each channel type and presence or absence of peptide/Trisk 95 is measured from 90 s of activity at both +40 mV and −40 mV (pooled). The fold change is the Po value after trans addition of Triadin peptide or full length Trisk 95, relative to Po in the absence of Triadin peptide/Trisk 95. Trisk 95 was isolated from rabbit skeletal muscle and these data (in italics) have been previously published in [13] and are included here for comparison. Significant (p≤0.05) differences in Po upon addition of Triadin peptide or Trisk 95 are indicated for each condition compared to its control (*).

  • Triadin peptide modulates purified RyR1 single channel open time, closed time and closed frequency.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A) Average data for open probability (Po) in presence of Triadin200–232 and Triadin200–231 (n = 10), data from both peptides combined (see results text, collectively termed Triadin peptide, n = 11–20) for each of the following parameters; open time (To), close time (Tc) and open frequency (Fo), collected at −40 mV and +40 mV. All data is expressed as relative mean data (Log rel (parameter)). Relative mean Po (log rel Po) is the average of differences between the log10 of the Po in the presence of either 63 nM or 252 nM Triadin peptide (log10 PoPep) and log10 of the control Po (log10 PoCon) for each channel. Log rel To is log10 ToPep-log10ToCon, log rel Tc is log10 TcPep-log10 TcCon and log rel Fo is log10 FoPep-log10 FoCon. (B) [3H]ryanodine binding to purified RyR1 in the absence and presence of 63 nM of Triadin peptide. [3H]ryanodine binding is measured as pmol ryanodine/mg RyR1. Data is expressed relative to binding recorded in the absence of peptides (rel ryanodine binding). Significance (p≤0.05) is indicated for each concentration compared to activity recorded prior to addition of peptide (*).

  • Triadin peptides modulate purified RyR1.
    2013
    Co-Authors: Elize Wium, Angela F. Dulhunty, Nicole A. Beard
    Abstract:

    (A) Running histogram of a typical bilayer experiment. Open probability (Po) was measured every 30 s throughout the lifetime of the experiment before and after the addition of 63 nM and then 252 nM Triadin200–231 peptide (arrows) at +40 mV (light grey bins) and −40 mV (dark grey bins). Data averages for each condition are shown as horizontal broken lines for +40 mV and −40 mV, and median is presented as a horizontal solid line. (B, C) 3 s traces of purified RyR1 channel activity. Activity was recorded at +40 mV (left) where channels are opening upward from zero current (c, continuous line) to maximum open conductance (o, broken line) and at −40 mV (right), where channel openings are downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). Top panel – control recording of purified RyR1 prior to the addition of Triadin peptide; middle and bottom panel – after the addition of 63 nM and 252 nM Triadin peptide to the trans chamber. (B) Shows addition of Triadin200–232 peptide, and (C) shows addition of Triadin200–231. Control Po in the absence of peptide was 0.46±0.12 at +40 mV and 0.38±0.17 at −40 mV (for Triadin200–232), and 0.14±0.07 at +40 mV and 0.29±0.17 at −40 mV (for Triadin200–231).

Isabelle Marty - One of the best experts on this subject based on the ideXlab platform.

Julie Brocard - One of the best experts on this subject based on the ideXlab platform.

  • Additional file 8: of Deletion of the microtubule-associated protein 6 (MAP6) results in skeletal muscle dysfunction
    2018
    Co-Authors: Muriel Sébastien, Julie Brocard, Benoit Giannesini, Perrine Aubin, Mathilde Chivet, Laura Pietrangelo, Simona Boncompagni, Christophe Bosc, Jacques Brocard, John Rendu
    Abstract:

    Figure S6. Organization of triads and microtubules is similar in WT and MAP6 KO myotubes. WT and MAP6 primary cultures were differentiated for 3 days before being fixed and labeled with antibodies against Triadin and RyR1 to visualize the triads and against tubulin to visualize the microtubules. No major difference is observed between the two genotypes for Triadin, RyR, and tubulin. (JPG 1165 kb

  • Author manuscript, published in "The Journal of Biological Chemistry 2005;280(31):28601-9" DOI: 10.1074/jbc.M501484200 TriadinS ARE NOT TRIAD SPECIFIC PROTEINS: TWO NEW SKELETAL MUSCLE TriadinS POSSIBLY INVOLVED IN THE ARCHITECTURE OF SARCOPLASMIC RETICUL
    2013
    Co-Authors: Stéphane Vassilopoulos, Dominique Thevenon, Sophia Smida Rezgui, Julie Brocard, Alain Lacampagne, Joël Lunardi, Michel Dewaard, Isabelle Marty
    Abstract:

    We have cloned two new Triadin isoforms from rat skeletal muscle, Trisk 49 and Trisk 32, named according to their theoretical molecular weights, 49 kDa and 32 kDa respectively. Specific antibodies directed against each protein were produced to characterize both new Triadins. Both are expressed in adult rat skeletal muscle, and their expression in slow twitch muscle is lower than in fast twitch muscle. Using double immunofluorescent labeling, the localization of these two Triadins was studied in comparison to well characterized proteins such as ryanodine receptor, calsequestrin, desmin, Ca 2+-ATPase, titin. None of these two Triadins are localized within the rat skeletal muscl

  • absence of Triadin a protein of the calcium release complex is responsible for cardiac arrhythmia with sudden death in human
    Human Molecular Genetics, 2012
    Co-Authors: Nathalie Rouxbuisson, Julie Brocard, Marine Cacheux, Anne Fourestlieuvin, Jeremy Fauconnier, Isabelle Denjoy, Philippe Durand
    Abstract:

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on Triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the Triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in Triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant Triadin in Triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of Triadin for the normal function of the cardiac calcium release complex in humans.

  • Nathalie Roux-Buisson1,2,3,4,{, Marine Cacheux1,4,{, Anne Fourest-Lieuvin1,4,5,
    2012
    Co-Authors: Jeremy Fauconnier, Julie Brocard, Isabelle Denjoy, Pascale Guicheney Florence Kyndt, Isabelle Marty
    Abstract:

    Absence of Triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmi

  • INSERM U836 / Eq Muscle et Pathologies UJF Site Santé
    2010
    Co-Authors: Stéphane Vassilopoulos, Julie Brocard, Julien Fauré, Anne Fourest-lieuvin, Sarah Oddoux, Isabelle Marty
    Abstract:

    During the last 20 years, the identification of Triadin function in cardiac and skeletal muscle has been the focus of numerous studies. First thought as the missing link between the ryanodine receptor and the dihydropyridines receptor and responsible of skeletal type excitation-contraction coupling, the current hypothesis on Triadin function has slowly evolved, and Triadin is envisaged now as a regulator of calcium release, both in cardiac and skeletal muscle. Nevertheless, none of the experiments performed up to now gave a clear cut view of what Triadin really does in muscle. The problem became more complex with the identification of multiple Triadin isoforms, having possibly multiple functions. Using a different approach from what has been done previously, we obtained new clues about the function of Triadin. Our data point to a possible involvement of Triadin in reticulum structure, in relation with the microtubule network

Larry R Jones - One of the best experts on this subject based on the ideXlab platform.

  • Altered function in atrium of transgenic mice overexpressing Triadin 1. Am J Physiol Heart Circ Physiol. 2002;283:H1334–H1343. doi: 10.1152/ajpheart.00937.2001
    2016
    Co-Authors: Uwe Kirchhefer, Larry R Jones, Yvonne M. Kobayashi, Hideo A. Baba, Wilhelm Schmitz, Joachim Neumann, Yvonne M. Koba
    Abstract:

    10.1152/ajpheart.00937.2001.—Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, pro-teins that are important for Ca2 release. To better under-stand the role of Triadin 1 in SR-Ca2 release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac Triadin 1 under control of the -myosin heavy chain (MHC) promoter. Three-week-old transgenic atria ex-hibited mild hypertrophy. Finally, atrial weight was in-creased by 110 % in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR Ca2-ATPase isoform. Force of contraction was already decreased in 3-wk-old trans

  • Triadins modulate intracellular ca2 homeostasis but are not essential for excitation contraction coupling in skeletal muscle
    Journal of Biological Chemistry, 2007
    Co-Authors: Xiaohua Shen, Larry R Jones, Yvonne M. Kobayashi, Clara Franziniarmstrong, Jose R Lopez, Y Wang, Glenn W L Kerrick, Anthony H Caswell, James D Potter
    Abstract:

    To unmask the role of Triadin in skeletal muscle we engineered pan-Triadin-null mice by removing the first exon of the Triadin gene. This resulted in a total lack of Triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of Triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca2+ imaging studies in null lumbricalis muscles and myotubes showed that the lack of Triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+.[3H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ inhibition between wild-type and Triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for Triadin in regulating myoplasmic Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

  • the role of calsequestrin Triadin and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium
    Biophysical Journal, 2004
    Co-Authors: Inna Gyorke, Larry R Jones, Nichole Hester, Sandor Gyorke
    Abstract:

    The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), Triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 μM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When Triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with Triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, Triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas Triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.

  • complex formation between junctin Triadin calsequestrin and the ryanodine receptor proteins of the cardiac junctional sarcoplasmic reticulum membrane
    Journal of Biological Chemistry, 1997
    Co-Authors: Lin Zhang, Yvonne M. Kobayashi, Jeff Kelley, Glen Schmeisser, Larry R Jones
    Abstract:

    Several key proteins have been localized to junctional sarcoplasmic reticulum which are important for Ca2+ release. These include the ryanodine receptor, Triadin, and calsequestrin, which may associate into a stable complex at the junctional membrane. We recently purified and cloned a fourth component of this complex, junctin, which exhibits homology with Triadin and is the major 125I-calsequestrin-binding protein detected in cardiac sarcoplasmic reticulum vesicles (Jones, L. R., Zhang, L., Sanborn, K., Jorgensen, A. O., and Kelley, J. (1995) J. Biol. Chem. 270, 30787-30796). In the present study, we have examined the binding interactions between the cardiac forms of these four proteins with emphasis placed on the role of junctin. By a combination of approaches including calsequestrin-affinity chromatography, filter overlay, immunoprecipitation assays, and fusion protein binding analyses, we find that junctin binds directly to calsequestrin, Triadin, and the ryanodine receptor. This binding interaction is localized to the lumenal domain of junctin, which is highly enriched in charged amino acids organized into "KEKE" motifs. KEKE repeats are also found in the common lumenal domain of Triadin, which likewise is capable of binding to calsequestrin and the ryanodine receptor (Guo, W., and Campbell, K. P. (1995) J. Biol. Chem. 270, 9027-9030). It appears that junctin and Triadin interact directly in the junctional sarcoplasmic reticulum membrane and stabilize a complex that anchors calsequestrin to the ryanodine receptor. Taken together, these results suggest that junctin, calsequestrin, Triadin, and the ryanodine receptor form a quaternary complex that may be required for normal operation of Ca2+ release.

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