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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling</title><author><name sortKey="Goonasekera, Sanjeewa A" sort="Goonasekera, Sanjeewa A" uniqKey="Goonasekera S" first="Sanjeewa A." last="Goonasekera">Sanjeewa A. Goonasekera</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Beard, Nicole A" sort="Beard, Nicole A" uniqKey="Beard N" first="Nicole A." last="Beard">Nicole A. Beard</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Groom, Linda" sort="Groom, Linda" uniqKey="Groom L" first="Linda" last="Groom">Linda Groom</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Kimura, Takashi" sort="Kimura, Takashi" uniqKey="Kimura T" first="Takashi" last="Kimura">Takashi Kimura</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Lyfenko, Alla D" sort="Lyfenko, Alla D" uniqKey="Lyfenko A" first="Alla D." last="Lyfenko">Alla D. Lyfenko</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Rosenfeld, Andrew" sort="Rosenfeld, Andrew" uniqKey="Rosenfeld A" first="Andrew" last="Rosenfeld">Andrew Rosenfeld</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Marty, Isabelle" sort="Marty, Isabelle" uniqKey="Marty I" first="Isabelle" last="Marty">Isabelle Marty</name><affiliation><nlm:aff id="aff3">INSERM U607; CEA Grenoble, DRDC, F38054 Grenoble cedex, France</nlm:aff></affiliation></author><author><name sortKey="Dulhunty, Angela F" sort="Dulhunty, Angela F" uniqKey="Dulhunty A" first="Angela F." last="Dulhunty">Angela F. Dulhunty</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Dirksen, Robert T" sort="Dirksen, Robert T" uniqKey="Dirksen R" first="Robert T." last="Dirksen">Robert T. Dirksen</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">17846166</idno><idno type="pmc">2151650</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151650</idno><idno type="RBID">PMC:2151650</idno><idno type="doi">10.1085/jgp.200709790</idno><date when="2007">2007</date><idno type="wicri:Area/Pmc/Corpus">001129</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001129</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling</title><author><name sortKey="Goonasekera, Sanjeewa A" sort="Goonasekera, Sanjeewa A" uniqKey="Goonasekera S" first="Sanjeewa A." last="Goonasekera">Sanjeewa A. Goonasekera</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Beard, Nicole A" sort="Beard, Nicole A" uniqKey="Beard N" first="Nicole A." last="Beard">Nicole A. Beard</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Groom, Linda" sort="Groom, Linda" uniqKey="Groom L" first="Linda" last="Groom">Linda Groom</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Kimura, Takashi" sort="Kimura, Takashi" uniqKey="Kimura T" first="Takashi" last="Kimura">Takashi Kimura</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Lyfenko, Alla D" sort="Lyfenko, Alla D" uniqKey="Lyfenko A" first="Alla D." last="Lyfenko">Alla D. Lyfenko</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Rosenfeld, Andrew" sort="Rosenfeld, Andrew" uniqKey="Rosenfeld A" first="Andrew" last="Rosenfeld">Andrew Rosenfeld</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author><author><name sortKey="Marty, Isabelle" sort="Marty, Isabelle" uniqKey="Marty I" first="Isabelle" last="Marty">Isabelle Marty</name><affiliation><nlm:aff id="aff3">INSERM U607; CEA Grenoble, DRDC, F38054 Grenoble cedex, France</nlm:aff></affiliation></author><author><name sortKey="Dulhunty, Angela F" sort="Dulhunty, Angela F" uniqKey="Dulhunty A" first="Angela F." last="Dulhunty">Angela F. Dulhunty</name><affiliation><nlm:aff id="aff2">Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</nlm:aff></affiliation></author><author><name sortKey="Dirksen, Robert T" sort="Dirksen, Robert T" uniqKey="Dirksen R" first="Robert T." last="Dirksen">Robert T. Dirksen</name><affiliation><nlm:aff id="aff1">Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of General Physiology</title><idno type="ISSN">0022-1295</idno><idno type="eISSN">1540-7748</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Ca<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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 and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca<sup>2+</sup> release during excitation–contraction coupling in skeletal muscle.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Gen Physiol</journal-id><journal-title>The Journal of General Physiology</journal-title><issn pub-type="ppub">0022-1295</issn><issn pub-type="epub">1540-7748</issn><publisher><publisher-name>The Rockefeller University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">17846166</article-id><article-id pub-id-type="pmc">2151650</article-id><article-id pub-id-type="publisher-id">200709790</article-id><article-id pub-id-type="doi">10.1085/jgp.200709790</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject><subj-group><subject>Article</subject></subj-group></subj-group></article-categories><title-group><article-title>Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Goonasekera</surname><given-names>Sanjeewa A.</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Beard</surname><given-names>Nicole A.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Groom</surname><given-names>Linda</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kimura</surname><given-names>Takashi</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lyfenko</surname><given-names>Alla D.</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Rosenfeld</surname><given-names>Andrew</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Marty</surname><given-names>Isabelle</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Dulhunty</surname><given-names>Angela F.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Dirksen</surname><given-names>Robert T.</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642</aff><aff id="aff2"><label>2</label>Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT, 2601, Australia</aff><aff id="aff3"><label>3</label>INSERM U607; CEA Grenoble, DRDC, F38054 Grenoble cedex, France</aff><author-notes><fn><p>Correspondence to Robert T. Dirksen: <email>Robert_Dirksen@URMC.Rochester.edu</email></p></fn></author-notes><pub-date pub-type="ppub"><month>10</month><year>2007</year></pub-date><volume>130</volume><issue>4</issue><fpage>365</fpage><lpage>378</lpage><history><date date-type="received"><day>23</day><month>3</month><year>2007</year></date><date date-type="accepted"><day>22</day><month>8</month><year>2007</year></date></history><permissions><copyright-statement>Copyright © 2007, The Rockefeller University Press</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="jgp1300365.pdf"></self-uri><abstract><p>Ca<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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<sup>2+</sup> 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 and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca<sup>2+</sup> release during excitation–contraction coupling in skeletal muscle.</p></abstract></article-meta><notes><fn-group><fn><p>S.A. Goonasekera and N.A. Beard contributed equally to this work.</p></fn><fn><p>Abbreviations used in this paper: 4-cmc, 4-chloro-m-cresol; CSQ, calsequestrin; DHPR, dihydropyridine receptor; EC, excitation-contraction; RyR1, ryanodine receptor type-1; WT, wild type.</p></fn></fn-group></notes></front><body><sec><title>INTRODUCTION</title><p>Ca<sup>2+</sup> signaling in most cells depends on Ca<sup>2+</sup> release from intracellular stores. The efficiency of Ca<sup>2+</sup> release is determined by the Ca<sup>2+</sup> binding capacity of the store proteins and the activity of Ca<sup>2+</sup> release channels in the store membrane. The Ca<sup>2+</sup> store in striated muscle, the SR, plays a central role in the vital functions of movement, respiration, and heart beat (<xref ref-type="bibr" rid="bib31">Rossi and Dirksen, 2006</xref>). The key Ca<sup>2+</sup> binding protein in the SR is calsequestrin (CSQ) and the Ca<sup>2+</sup> release channel is the ryanodine receptor (RyR) (<xref ref-type="bibr" rid="bib35">Zhang et al., 1997</xref>; <xref ref-type="bibr" rid="bib4">Beard et al., 2004</xref>). CSQ not only binds Ca<sup>2+</sup> but also regulates Ca<sup>2+</sup> release by communicating with the RyR via two intermediary proteins, triadin and junctin (<xref ref-type="bibr" rid="bib3">Beard et al., 2002</xref>; <xref ref-type="bibr" rid="bib13">Gyorke et al., 2004</xref>; <xref ref-type="bibr" rid="bib34">Wei et al., 2006</xref>), which are found in many tissues and play a ubiquitous role in Ca<sup>2+</sup> signaling. Both are transmembrane proteins that bind to CSQ and the RyR (<xref ref-type="bibr" rid="bib16">Jones et al., 1995</xref>) to form a CSQ/triadin/junctin/RyR “luminal Ca<sup>2+</sup> transduction machine” that is central to Ca<sup>2+</sup> release unit function (<xref ref-type="bibr" rid="bib3">Beard et al., 2002</xref>, <xref ref-type="bibr" rid="bib4">2004</xref>; <xref ref-type="bibr" rid="bib13">Gyorke et al., 2004</xref>; <xref ref-type="bibr" rid="bib34">Wei et al., 2006</xref>) and assembly (<xref ref-type="bibr" rid="bib33">Tijskens et al., 2003</xref>).</p><p>Triadin is a junctional SR protein discovered in 1990 by <xref ref-type="bibr" rid="bib6">Brandt et al. (1990)</xref> that was originally proposed to play a critical role in excitation–contraction (EC) coupling (<xref ref-type="bibr" rid="bib17">Kim et al., 1990</xref>). However, since the bulk of the protein is located within the SR lumen where it binds to CSQ and the RyR (<xref ref-type="bibr" rid="bib22">Knudson et al., 1993b</xref>; <xref ref-type="bibr" rid="bib12">Guo and Campbell, 1995</xref>), it is currently believed to facilitate cross-talk between CSQ and the RyR (<xref ref-type="bibr" rid="bib3">Beard et al., 2002</xref>), rather than directly influence EC coupling (<xref ref-type="bibr" rid="bib13">Gyorke et al., 2004</xref>). Junctin was later discovered and thought to have a comparable function to triadin, due to its similar structure and ability to also bind CSQ and the RyR (<xref ref-type="bibr" rid="bib16">Jones et al., 1995</xref>; <xref ref-type="bibr" rid="bib35">Zhang et al., 1997</xref>; <xref ref-type="bibr" rid="bib33">Tijskens et al., 2003</xref>). The specific residues in RyR1 that bind triadin and junctin are functionally relevant and, therefore, of great interest. A putative triadin binding site was recently identified in the terminal intraluminal loop of RyR1 between residues 4860 and 4917 (<xref ref-type="bibr" rid="bib25">Lee et al., 2004</xref>). More recently, alanine substitution of three specific negatively charged residues within this region (D4878, D4907, and E4908) was found to disrupt triadin binding to full-length RyR1 and also alter the magnitude and kinetics of caffeine-induced Ca<sup>2+</sup> release (<xref ref-type="bibr" rid="bib24">Lee et al., 2006</xref>). However, the relative impact of disrupting the triadin–RyR1 interaction on Ca<sup>2+</sup> release during EC coupling, potential direct effects of the mutations on RyR1 function that are unrelated to triadin binding, or effects of the mutations on junctin binding to RyR1 have not been investigated.</p><p>Here we demonstrate that the intraluminal RyR1 residues D4878, D4907, and E4908 contribute unequally (D4907 > E4908 > D4878) to triadin binding to RyR1 and that this interaction is an important regulator of both voltage- and ligand-induced SR Ca<sup>2+</sup> release in skeletal muscle. Moreover, we found that disruption of triadin binding to RyR1 did not affect junctional targeting of RyR1, RyR1 enhancement of dihydropyridine receptor (DHPR) calcium channel activity (retrograde coupling; see <xref ref-type="bibr" rid="bib7">Dirksen, 2002</xref> for review), or the ability of junctin to bind to RyR1. We propose a model whereby triadin binding to RyR1 enhances release channel opening in response to both voltage- and ligand-induced activation and that this activity is important for ensuring robust and rapid calcium release during EC coupling.</p></sec><sec sec-type="materials|methods"><title>MATERIALS AND METHODS</title><sec><title>Preparation of Mutants and Nuclear Microinjection of Dyspedic Myotubes</title><p>The D4878A (ΔM<sub>1</sub>), D4907A (ΔM<sub>2</sub>), E4908A (ΔM<sub>3</sub>), D4878A/D4907A (ΔM<sub>1,2</sub>), D4878A/E4908A (ΔM<sub>1,3</sub>), D4907A/E4908A (ΔM<sub>2,3</sub>), and D4878A/D4907A/E4908A (ΔM<sub>1,2,3</sub>) mutations were introduced into a full-length rabbit RyR1 cDNA using standard two-step site-directed mutagenesis. All sequences generated and modified by PCR were checked for integrity by sequence analysis. Primary cultures of dyspedic myotubes were cultured from skeletal myoblasts isolated from newborn dyspedic mice (<xref ref-type="bibr" rid="bib27">Nakai et al., 1996</xref>; <xref ref-type="bibr" rid="bib1">Avila et al., 2001</xref>). After allowing myoblasts to differentiate into multinucleated myotubes for 4–7 d, nuclei of individual myotubes were microinjected with cDNAs encoding CD8 (0.1 μg/μl) and one of the RyR1 constructs at a concentration of 0.5 μg/μl. Expressing myotubes were subsequently identified 48–72 h after injection by incubation with CD8 antibody-coated beads (Dynabeads, Dynal ASA). All animals were anaesthetized and humanely killed following procedures that were reviewed and approved by the University Committee on Animals Resources at the University of Rochester School of Medicine and Dentistry.</p></sec><sec><title>Measurements of Electrically Evoked and Agonist-induced Ca<sup>2+</sup> Transients in Myotubes</title><p>Intracellular Ca<sup>2+</sup> measurements in intact myotubes were obtained from Indo-1 AM (TefLabs Inc.) loaded myotubes (<xref ref-type="bibr" rid="bib1">Avila et al., 2001</xref>). Cytosolic dye within a rectangular region of the cell was excited at 350 nm and fluorescence emission at 405 and 485 nm was measured at 100 Hz sampling frequency using a 40× oil objective, a photomultiplier detection system (Photon Technology International), and results are presented as the ratio of 405 and 485 nm (F<sub>405</sub>/F<sub>485</sub>). These Ca<sup>2+</sup> measurements were conducted in a normal rodent Ringer's solution consisting of (in mM) 145 NaCl, 5 KCl, 2 CaCl<sub>2</sub>, 1 MgCl<sub>2</sub>, 10 HEPES, pH 7.4. Electrically evoked Ca<sup>2+</sup> transients were elicited using field stimulation (8 V, 20 ms) applied every 10 s. A caffeine (30 mM) or 4-chloro-m- cresol (4-cmc, 500 μM) bolus was delivered at the end of the protocol to assess release channel expression and SR Ca<sup>2+</sup> content. Data are presented as mean ± SEM with significance accepted at P < 0.01 (unpaired Student's <italic>t</italic> test).</p></sec><sec><title>Simultaneous Measurements of Macroscopic Ca<sup>2+</sup> Currents and Transients in Myotubes</title><p>Whole-cell patch clamp experiments were used to simultaneously measure voltage-gated L-type Ca<sup>2+</sup> currents and Ca<sup>2+</sup> transients (<xref ref-type="bibr" rid="bib1">Avila et al., 2001</xref>). Peak L-current magnitude was normalized to cell capacitance (pA/pF) and plotted as a function of membrane potential (V<sub>m</sub>) and fitted according to <disp-formula id="fd1"><label>(1)</label><tex-math id="M1">\documentclass[10pt]{article}
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\begin{equation*}{\mathrm{I}}={\mathrm{G}}_{{\mathrm{max}}}({\mathrm{V}}_{{\mathrm{m}}}-{\mathrm{V}}_{{\mathrm{rev}}})/(1+{\mathrm{exp}}[({\mathrm{V}}_{{\mathrm{G}}1/2}-{\mathrm{V}}_{{\mathrm{m}}})/{\mathrm{k}}_{{\mathrm{G}}}]),\end{equation*}\end{document}</tex-math></disp-formula>where G<sub>max</sub> is the maximal L-channel conductance, V<sub>m</sub> is test potential, V<sub>rev</sub> is extrapolated reversal potential, V<sub>G1/2</sub> is the voltage for half-maximal activation of G<sub>max</sub>, and k<sub>G</sub> is a slope factor. Relative changes in intracellular Ca<sup>2+</sup> in these experiments were measured after dialysis with K<sub>5</sub>-Fluo-4 salt. Relative changes in Fluo-4 fluorescence (ΔF/F) were measured either 30 or 200 ms after depolarization, plotted as a function of V<sub>m</sub>, and fitted according to <disp-formula id="fd2"><label>(2)</label><tex-math id="M2">\documentclass[10pt]{article}
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\begin{equation*}{\mathrm{{\Delta}F/F}}=({\mathrm{{\Delta}F/F}}_{{\mathrm{max}}})/\{1+{\mathrm{exp}}[({\mathrm{V}}_{{\mathrm{F}}1/2}-{\mathrm{V}}_{{\mathrm{m}}})/{\mathrm{k}}_{{\mathrm{F}}}]\},\end{equation*}\end{document}</tex-math></disp-formula>where (ΔF/F)<sub>max</sub> is the calculated maximal change in fluorescence, V<sub>F1/2</sub> is the voltage for half-maximal activation of (ΔF/F)<sub>max</sub>, and k<sub>F</sub> is a slope factor. The bell-shaped voltage dependence of ΔF/F measurements obtained in ΔM<sub>1,2,3</sub>- and ΔM<sub>2,3</sub>-expressing myotubes was fitted according to the following equation: <disp-formula id="fd3"><label>(3)</label><tex-math id="M3">\documentclass[10pt]{article}
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\begin{equation*}{\mathrm{{\Delta}F/F}}=(({\mathrm{{\Delta}F/F}})_{{\mathrm{max}}}(({\mathrm{V}}_{{\mathrm{m}}}-{\mathrm{V}}_{{\mathrm{rev}}})/{\mathrm{k}}^{\prime}))/(1+{\mathrm{exp}}(({\mathrm{V}}_{{\mathrm{F}}1/2}-{\mathrm{V}}_{{\mathrm{m}}})/{\mathrm{k}}_{{\mathrm{F}}})),\end{equation*}\end{document}</tex-math></disp-formula>where (ΔF/F)<sub>max</sub>, V<sub>m</sub>, V<sub>rev</sub>, V<sub>F1/2</sub>, and k<sub>F</sub> have their usual meanings. The additional variable k' is a scaling factor that varies with (ΔF/F)<sub>max</sub> (<xref ref-type="bibr" rid="bib32">Sheridan et al., 2003</xref>; <xref ref-type="bibr" rid="bib9">Goonasekera et al., 2005</xref>). Pooled current–voltage (I-V) and fluorescence–voltage (ΔF/F-V) data in <xref ref-type="table" rid="tbl1">Table I</xref> are expressed as mean ± SEM. All patch clamp experiments were conducted in an external solution consisting of (in mM) 145 TEA-Cl, 10 CaCl<sub>2</sub>, and 10 HEPES (pH 7.4). The internal patch pipette solution consisted of (in mM) 145 Cs-Aspartate, 10 CsCl, 0.1 Cs<sub>2</sub>-EGTA, 1.2 MgCl<sub>2</sub>, 5 Mg-ATP, 0.2 K<sub>5</sub>-Fluo-4, and 10 HEPES (pH 7.4).</p></sec><sec><title>Measurements of 4-cmc-induced Ca<sup>2+</sup> Transients in HEK293 Cells</title><p>HEK293 cells plated on coverslips were transfected with either WT RyR1 or ΔM<sub>1,2,3</sub> cDNA (<xref ref-type="bibr" rid="bib15">Jiang et al., 2002</xref>). 2 d after transfection, cells were loaded with 5 μM Fura-2 AM for 30 min at 37°C in Ringer's solution. Coverslips of Fura-2–loaded cells were then mounted in a tissue chamber on the stage of an epifluorescence-equipped inverted microscope. Cells were sequentially excited at 340- and 380-nm wavelength and fluorescence emission at 510 nM was collected using a high-speed IMAGO-QE CCD camera (TILL Photonics). The results are presented as the ratio of F<sub>340</sub>/F<sub>380</sub> (collected at 6.25Hz). Maximal increase in intracellular Ca<sup>2+</sup> induced by addition of 4-cmc (500 μM) was defined as the difference between peak and baseline fluorescence ratios during agonist exposure.</p></sec><sec><title>Purification of Triadin and Junctin</title><p>Triadin and junctin were purified from rabbit skeletal muscle as previously described (<xref ref-type="bibr" rid="bib26">Mulvey and Ohlendieck, 2003</xref>) except that after separation by electrophoresis, proteins were eluted by gentle agitation at 37°C in a buffer containing 0.5% CHAPS, 20 mM MOPS, and 150 mM NaCl (pH 7.4). Proteins were then dialyzed twice against 20 mM MOPS, 150 mM NaCl, pH 7.4. Purification was enhanced by immunoselection with anti-junctin or anti-triadin.</p></sec><sec><title>[<sup>3</sup>H]Ryanodine Binding and Single Channel Recording of Purified RyRs</title><p>WT and ΔM<sub>1,2,3</sub> RyR1 constructs were expressed and purified from HEK293 cells as previously described (<xref ref-type="bibr" rid="bib18">Kimura et al., 2005</xref>; <xref ref-type="bibr" rid="bib19">Kimura et al., 2007</xref>). [<sup>3</sup>H]Ryanodine binding was performed as previously described (<xref ref-type="bibr" rid="bib18">Kimura et al., 2005</xref>), except that 5 mg of purified RyR1 protein was used in all experiments and in some experiments preincubation with triadin (5 μg/ml) was done for 30 min on ice, before initiating [<sup>3</sup>H]Ryanodine binding. Single channel experiments were conducted as described in (<xref ref-type="bibr" rid="bib18">Kimura et al., 2005</xref>), using the following conditions. To incorporate purified RyRs (WT or ΔM<sub>1,2,3</sub>), the standard incorporation solution consisted of the following: cis: 230 mM Cs-methanesulfonate, 20 mM CsCl, 5 mM CaCl<sub>2</sub>, and 10 mM TES (pH 7.4); and trans: 30 mM Cs-methanesulfonate, 20 mM CsCl, 1 mM CaCl<sub>2</sub>, and 10 mM TES (pH 7.4). Immediately after channel incorporation, 200 mM Cs-methanesulfonate was added to the trans chamber (to ensure recording solution symmetry) and cis Ca<sup>2+</sup> was adjusted to 1 mM, 10 μM, or 100 nM by the addition of Ca<sup>2+</sup> chelator (BAPTA). Channel activity was analyzed over 10–30-s periods of continuous activity at +40 and −40 mV. Slow fluctuations in the baseline were corrected using an in-house program written by Dr. D.R. Laver. Channel activity was measured either as “mean current” (average of all data points) or as open probability (P<sub>o</sub>), using threshold analysis with the program Channel 2, (developed by Drs. P.W. Gage and M. Smith, JCSMR). Measurements of mean current, performed on 30-s records from bilayers containing one to four channels, included all channel activity from small subconductance openings to maximum levels. Mean current divided by the maximum current provides an approximate measure of P<sub>o</sub>. P<sub>o</sub>, mean open times (T<sub>o</sub>), and mean closed times (T<sub>c</sub>) were measured directly during periods of recording in which the opening of only a single channel was detected. Threshold levels for channel opening and closing were set at ∼20% of the maximum single channel conductance in order to exclude baseline noise.</p></sec><sec><title>Immunoprecipitations</title><p>Precleared purified skeletal muscle triadin or junctin were incubated with either anti-triadin or anti-junctin antibody coupled to protein A/G sepharose and incubated with purified recombinant RyR1s (expressed in HEK293 cells) for 2 h at 4°C in a buffer containing 20 mM MOPS, 150 mM NaCl, pH 7.4. Immunoprecipitates were washed three times in 20 mM MOPS, 150 mM NaCl (pH 7.4), proteins eluted from the beads by boiling for 2–3 min in Laemmli sample buffer, and subjected to SDS-PAGE and immunoblot. Identical results were obtained using similar buffers containing 1 mM Ca<sup>2+</sup>. Reverse immunoprecipitation was performed, using a sepharose/anti-RyR/RyR complex to assess binding of purified triadin and junctin to immobilized RyRs. Anti-RyR was obtained from Sigma-Aldrich. Each immunoprecipitation was repeated at least three times (<italic>n</italic> ≥ 3). Where appropriate, percent binding was calculated from three separate immunoprecipitates via immunoblot image analysis using the Genetools analysis software.</p></sec><sec><title>Immunofluorescence Labeling</title><p>Expressing dyspedic myotubes plated on glass coverslips were fixed and immunostained with a mouse monoclonal anti-RyR antibody (34C, 1:10; Developmental Studies Hybridoma Bank) and a sheep polyclonal anti-DHPR antibody (1:200; Upstate Biotechnology) overnight at 4°C as previously described (<xref ref-type="bibr" rid="bib10">Graves and Hinkle, 2003</xref>). On the following day, coverslips were washed with PBS three times each for 5 min and then incubated for 1 h at room temperature in blocking buffer containing a 1:500 dilution of Alexa Fluor 488–labeled donkey anti-mouse IgG (Molecular Probes) and 1:500 dilution of rhodamine-labeled donkey anti-sheep IgG (Jackson ImmunoResearch Laboratories Inc.) and washed with PBS (three times for 5 min each). Coverslips were mounted on glass slides and images obtained using a Nikon Eclipse-C1 confocal microscope (Nikon Instruments Inc.) and a 40× oil objective. All confocal images were sampled at a spatial resolution (pixel diameter) of 100 nm. A similar protocol was used to immunostain and obtain images from WT RyR1- and ΔM<sub>1,2,3</sub>-expressing HEK293 cells using antibody 34C (1:20) and a rhodamine-conjugated goat anti-mouse IgG (1:2,000).</p></sec></sec><sec><title>RESULTS</title><sec><title>Three Negatively Charged Residues in the C-terminal Intraluminal Loop of RyR1 Unequally Coordinate Triadin Binding</title><p>To determine the relative importance of the three negatively charged RyR1 residues identified by <xref ref-type="bibr" rid="bib25">Lee et al. (2004)</xref> (<xref rid="fig1" ref-type="fig">Fig. 1 A</xref>) for triadin and junctin binding to full-length RyR1, we assessed the effects of a comprehensive series of mutations to these residues in triadin and junctin coimmunoprecipitation. Purified skeletal triadin or junctin were coupled to protein A/G sepharose via anti-triadin or anti-junctin antibodies (respectively), and association with WT and mutated RyR1 constructs was determined by incubation with purified recombinant RyR1 constructs. In contrast to <xref ref-type="bibr" rid="bib25">Lee et al. (2004)</xref>, triadin binding to RyR1 was not altered by any of the single mutants (D4878A, ΔM<sub>1</sub>; D4907A, ΔM<sub>2</sub>; E4908A, ΔM<sub>3</sub>) or for D4878A/E4908A (ΔM<sub>1,3</sub>) (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>), with similar amounts of each mutant binding to triadin as that observed for WT RyR1. However, a 50% ± 12% (<italic>n</italic> = 3) reduction in triadin binding was observed for D4878A/D4907A (ΔM<sub>1</sub>,<sub>2</sub>) while triadin binding to D4907A/E4908A (ΔM<sub>2,3</sub>) and D4878A/D4907A/E4908A (ΔM<sub>1</sub>,<sub>2,3</sub>) was undetectable (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>). Identical results were obtained in the absence of calcium (as in <xref ref-type="bibr" rid="bib25">Lee et al., 2004</xref>) and in the presence of 1 mM Ca<sup>2+</sup>. In addition, identical results were also observed in reverse coimmunoprecipitation experiments in which purified WT RyR1 or ΔM<sub>1</sub>,<sub>2,3</sub> were coupled to protein A/G sepharose via anti-RyR and their ability to pull down purified triadin and junctin was detected (unpublished data). In marked contrast to triadin, none of the luminal loop mutations significantly altered the ability of RyR1 to interact with junctin (<xref rid="fig1" ref-type="fig">Fig. 1 C</xref>) since the amount of each RyR1 mutant construct associated with junctin was similar to that of WT RyR1.</p><fig position="float" id="fig1"><label>Figure 1.</label><caption><p>Triadin and junctin binding to RyR1 luminal loop mutants. (A) Proposed interaction between triadin and the terminal RyR1 luminal loop. (Top) Amino acid sequence for a portion of the luminal loop between the final two transmembrane domains of the rabbit RyR1 protein. Negatively charged amino acids mutated in this study (D4878, D4807, and E4809 on the left and M<sub>1</sub>, M<sub>2</sub>, and M<sub>3</sub> on the right in the schematic) are shown in italics. (Bottom) Representation of the triadin interaction with the terminal luminal loop of RyR1 is modified from <xref rid="fig5" ref-type="fig">Fig. 5</xref> of <xref ref-type="bibr" rid="bib25">Lee et al. (2004)</xref>. (B and C) Western blot analysis of proteins immunoprecipitated with anti-triadin (B) or anti-junctin (C). After incubation of the WT or mutant RyR1 (listed at the bottom of the blot) with either triadin (B) or junctin (C). Immunoprecipitations were performed and the immunoprecipitated protein analyzed by Western Blot using anti-RyR and anti-triadin (B) or anti-junctin (C).</p></caption><graphic xlink:href="jgp1300365f01"></graphic></fig><p>Our results in <xref rid="fig1" ref-type="fig">Fig. 1</xref> were not influenced by the use of purified proteins. In initial experiments, solubilized SR was used as a source of triadin to pull down WT or ΔM<sub>1</sub>,<sub>2,3</sub> RyR1. The SR was exposed to 10 mM Ca<sup>2+</sup> to inhibit the interaction between CSQ and either triadin or junctin (<xref ref-type="bibr" rid="bib5">Beard et al., 2005</xref>) and to minimize interactions between triadin and junctin (<xref ref-type="bibr" rid="bib35">Zhang et al., 1997</xref>), before exposure to anti-triadin protein A/G sepharose. We found that exposure to 10 mM Ca<sup>2+</sup> also dissociated triadin from the RyR. When the Ca<sup>2+</sup> concentration was lowered appropriately, the anti-triadin/native triadin complex pulled down WT RyR1, but not ΔM<sub>1,2,3</sub> RyR1, as was observed using purified triadin in <xref rid="fig1" ref-type="fig">Fig. 1</xref>. Interestingly, in the light of experiments reported by <xref ref-type="bibr" rid="bib24">Lee et al. (2006)</xref>, we also performed experiments using anti-triadin antibody to pull down triadin from solubilized SR vesicles isolated from adult rabbit SR with either 0 or 1 mM Ca<sup>2+</sup> and found that the anti-triadin antibody pulled down a suite of proteins that included the RyR, triadin, CSQ, junctin, histidine-rich protein, and several other unidentified proteins.</p></sec><sec><title>The Triadin Binding-deficient RyR1 Mutant (ΔM<sub>1,2,3</sub>) Lacks Electrically Evoked Ca<sup>2+</sup> Release and Exhibits Slowed Kinetics of Agonist-induced Ca<sup>2+</sup> Release</title><p>To determine the consequences of disrupting triadin binding to RyR1 on skeletal muscle EC coupling, we expressed either WT RyR1 or ΔM<sub>1,2,3</sub> in myotubes derived from RyR1-null (dyspedic) mice. Extracellular electrical stimulation (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, closed triangles) evoked robust and rapid global Ca<sup>2+</sup> transients in intact Indo-1 AM–loaded WT RyR1-expressing myotubes (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, left), but not in uninjected (naive) dyspedic myotubes (unpublished data). However, while average resting Indo-1 ratios were not significantly different (resting F<sub>405</sub>/F<sub>485</sub> was 0.60 ± 0.01, <italic>n</italic> = 28, and 0.56 ± 0.01, <italic>n</italic> = 27, for WT RyR1- and ΔM<sub>1,2,3</sub>-expressing myotubes, respectively), ΔM<sub>1,2,3</sub>-expressing myotubes lacked electrically evoked Ca<sup>2+</sup> transients (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, right, and <xref rid="fig2" ref-type="fig">Fig. 2 B</xref>). To confirm functional ΔM<sub>1,2,3</sub> expression, a maximal concentration (30 mM) of caffeine or 4-cmc (500μM) was applied to both WT- and ΔM<sub>1,2,3</sub>-expressing myotubes (<xref rid="fig2" ref-type="fig">Fig. 2, A and B</xref>). Ca<sup>2+</sup> release in response to caffeine was reduced in ΔM<sub>1,2,3</sub>-expressing myotubes (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>) and the kinetics of caffeine-induced and 4-cmc–induced Ca<sup>2+</sup> release (time to peak and <italic>t</italic><sub>1/2</sub> of decay) were significantly slowed (<xref rid="fig2" ref-type="fig">Fig. 2, A and C</xref>).</p><fig position="float" id="fig2"><label>Figure 2.</label><caption><p>Effects of ΔM<sub>1,2,3</sub> on ligand-induced Ca<sup>2+</sup> release. (A) Representative indo-1 ratio traces obtained from intact dyspedic myotubes expressing either WT RyR1 (left) or ΔM<sub>1,2,3</sub> (right) following electrical stimulation (filled triangles) and caffeine application (horizontal bars). (B) Average maximal magnitude of electrically evoked (left), caffeine-induced (middle), and 4-cmc–induced (right) Ca<sup>2+</sup> release. (C) Average time to peak (TTP, left) and <italic>t</italic><sub>1/2</sub> of decay (middle) for caffeine-induced Ca<sup>2+</sup> release and time to peak 4-cmc Ca<sup>2+</sup> release (TTP, right). *, P < 0.01.</p></caption><graphic xlink:href="jgp1300365f02"></graphic></fig></sec><sec><title>The Triadin Binding-deficient RyR1 Mutant (ΔM<sub>1,2,3</sub>) Supports Retrograde, but not Orthograde, DHPR–RyR1 Coupling</title><p>Simultaneous whole-cell patch clamp measurements of voltage-gated L-type Ca<sup>2+</sup> currents and intracellular Ca<sup>2+</sup> transients were used to determine the ability of the ΔM<sub>1,2,3</sub> triadin binding-deficient mutant to support the retrograde and orthograde signals of skeletal muscle EC coupling (<xref rid="fig3" ref-type="fig">Fig. 3 A</xref>). Naive dyspedic myotubes exhibited small (<1 pA/pF) L-type Ca<sup>2+</sup> currents (<xref rid="fig3" ref-type="fig">Fig. 3 C</xref>, open squares) and lacked voltage-gated Ca<sup>2+</sup> release (<xref rid="fig3" ref-type="fig">Fig. 3 D</xref>, open squares) (<xref ref-type="bibr" rid="bib27">Nakai et al., 1996</xref>). Expression of WT RyR1 (<xref rid="fig3" ref-type="fig">Fig. 3 A</xref>) restored robust (∼10 pA/pF) voltage-gated L-type Ca<sup>2+</sup> currents (bottom traces) and Ca<sup>2+</sup> transients (top traces). Interestingly, while expression of ΔM<sub>1,2,3</sub> (<xref rid="fig3" ref-type="fig">Fig. 3 B</xref>) similarly enhanced L-type Ca<sup>2+</sup> current density, Ca<sup>2+</sup> release was markedly slower and reduced in magnitude. Voltage-gated Ca<sup>2+</sup> transients in WT RyR1-expressing myotubes exhibited the characteristic sigmoidal voltage dependence of skeletal muscle EC coupling (<xref rid="fig3" ref-type="fig">Fig. 3 D</xref>, filled circles), while Ca<sup>2+</sup> transients in ΔM<sub>1,2,3</sub>-expressing myotubes exhibited a bell-shaped voltage dependence (<xref rid="fig3" ref-type="fig">Fig. 3 D</xref>, filled triangles) that mirrored that of the L-type Ca<sup>2+</sup> current (<xref rid="fig3" ref-type="fig">Fig. 3 C</xref>, filled triangles). The bell-shaped Ca<sup>2+</sup> transients in ΔM<sub>1,2,3</sub>-expressing myotubes largely arose from Ca<sup>2+</sup> influx–induced Ca<sup>2+</sup> release through ΔM<sub>1,2,3</sub> channels as they were markedly reduced by blockers of both the L-type Ca<sup>2+</sup> channel (0.5 mM Cd<sup>2+</sup> + 0.2 mM La<sup>3+</sup>, open symbols in <xref rid="fig3" ref-type="fig">Fig. 3 E</xref>) and the RyR (100μM ryanodine, open symbols in <xref rid="fig3" ref-type="fig">Fig. 3 F</xref>).</p><fig position="float" id="fig3"><label>Figure 3.</label><caption><p>Effects of ΔM<sub>1,2,3</sub> on orthograde and retrograde DHPR-RyR1 coupling. (A) Representative L-type Ca<sup>2+</sup> currents (bottom traces) and intracellular Ca<sup>2+</sup> transients (top traces) resulting from 200-ms depolarizations to −50, −10, +30, and +70 mV in a WT RyR1- expressing myotube. (B) Representative L-type Ca<sup>2+</sup> currents (bottom traces) and intracellular Ca<sup>2+</sup> transients (top traces) resulting from 200-ms depolarizations to −50, −10, +30, and +70 mV in a ΔM<sub>1,2,3</sub>-expressing myotube. (C and D) Average voltage dependence of peak L-type Ca<sup>2+</sup> current density (C) and intracellular Ca<sup>2+</sup> transients (D) in naive dyspedic myotubes (open squares), WT RyR1-expressing (closed circles), and ΔM<sub>1,2,3</sub>-expressing (closed triangles) myotubes. (E) Inhibition of L-type Ca<sup>2+</sup> currents (with 0.5 mM Cd<sup>2+</sup>/0.2 mM La<sup>3+</sup>, open symbols) markedly reduced (86 ± 7%, <italic>n</italic> = 5 at +30 mV) Ca<sup>2+</sup> transients in ΔM<sub>1,2,3</sub>-expressing myotubes (triangles) but only minimally reduced (16 ± 9%, <italic>n</italic> = 5 at +30 mV) Ca<sup>2+</sup> transients in WT RyR1-expressing myotubes (circles). (F) Blockade of Ca<sup>2+</sup> release with 100 μM ryanodine (open symbols) markedly reduced depolarization-induced Ca<sup>2+</sup> transients in both WT RyR1- (circles) and ΔM<sub>1,2,3</sub>-expressing (triangles) myotubes.</p></caption><graphic xlink:href="jgp1300365f03"></graphic></fig></sec><sec><title>RyR1 Mutations that Disrupt Triadin Binding Do Not Directly Alter Release Channel Function</title><p>The observed alterations in ligand and voltage-gated Ca<sup>2+</sup> release in ΔM<sub>1,2,3</sub>-expressing dyspedic myotubes documented in <xref rid="fig2" ref-type="fig">Figs. 2</xref> and <xref rid="fig3" ref-type="fig">3</xref> could either be due to effects of the mutations on triadin regulation of RyR1 or to direct effects of the mutations on channel function that are independent of triadin binding. To test for potential direct effects of the mutations, we compared the subcellular localization and function of WT and ΔM<sub>1,2,3</sub> RyR1 channels following expression in HEK293 cells (<xref rid="fig4" ref-type="fig">Figs. 4</xref> and <xref rid="fig5" ref-type="fig">5</xref>). WT and ΔM<sub>1,2,3</sub> channels exhibited identical reticulated ER localization (<xref rid="fig4" ref-type="fig">Fig. 4 A</xref>) following expression in HEK293 cells. Since HEK293 cells do not express triadin and 4-cmc activates Ca<sup>2+</sup> release through expressed RyR1 channels (<xref ref-type="bibr" rid="bib8">Fessenden et al., 2000</xref>), we compared 4-cmc–induced Ca<sup>2+</sup> release responses in WT- and ΔM<sub>1,2,3</sub>-expressing HEK293 cells. The magnitude and kinetics of 4-cmc–induced Ca<sup>2+</sup> release was not significantly different (P > 0.05) between WT- and ΔM<sub>1,2,3</sub>-expressing HEK293 cells (<xref rid="fig4" ref-type="fig">Fig. 4, B–D</xref>).</p><fig position="float" id="fig4"><label>Figure 4.</label><caption><p>Subcellular localization and Ca<sup>2+</sup> release function of WT RyR1 and ΔM<sub>1,2,3</sub> channels expressed in HEK293 cells. (A) Both WT RyR1 (left) and ΔM<sub>1,2,3</sub> (right) channels exhibit similar reticulated ER expression. (B) Representative fura-2 ratio (F<sub>340</sub>/F<sub>380</sub>) traces obtained from WT RyR1- (left) and ΔM<sub>1,2,3</sub>-expressing (right) HEK293 cells following addition of 500 μM 4-cmc (bar). Average peak (C) and time to peak (TTP) 4-cmc responses (D) obtained from WT RyR1- and ΔM<sub>1,2,3</sub>-expressing HEK293 cells.</p></caption><graphic xlink:href="jgp1300365f04"></graphic></fig><fig position="float" id="fig5"><label>Figure 5.</label><caption><p>Properties of purified WT RyR1 and ΔM<sub>1,2,3</sub> channels expressed in HEK293 cells. (A) Ca<sup>2+</sup> dependence of [<sup>3</sup>H]ryanodine binding (% maximum binding) to WT (closed circles) and ΔM<sub>1,2,3</sub> (closed squares) channels. (B) Effect of preincubation with 5 μg/ml purified triadin on [<sup>3</sup>H]ryanodine binding in the presence of either 100 nM or 1 mM Ca<sup>2+</sup> (<italic>n</italic> = 6 for each). *, P < 0.05. [<sup>3</sup>H]ryanodine binding to purified RyR1 in the presence of triadin (filled bars) is normalized to binding to purified RyR1 in absence of triadin (crosshatched bars). (C) Representative single channel records from artificial lipid bilayers incorporated with two purified WT (top) and two purified ΔM<sub>1,2,3</sub> (bottom) channels at −40 mV in the presence of 1 mM trans (luminal) Ca<sup>2+</sup> and 10 μM cis (cytoplasmic) Ca<sup>2+</sup>. The channels opened from the closed level (c) to either single open (o<sub>1</sub>) and double open (o<sub>2</sub>) levels. (D) Average data from four WT RyR1 channels and four ΔM<sub>1,2,3</sub> channels showing open probability measured as mean current (I<sub>mean</sub>) normalized to maximum current (I<sub>max</sub>) during periods in which only one or two channels were open in the bilayer. Open probability decreased when the cis Ca<sup>2+</sup> was increased from 10 μM to either 1 mM or 5 mM (data for 1 and 5 mM cis Ca<sup>2+</sup> at +40 and −40 mV were grouped in the average data). *, P < 0.05. (E) Periods of single channel activity in recordings from bilayers containing purified WT (top) and ΔM<sub>1,2,3</sub> (bottom) channels at +40 mV with 1 mM trans (luminal) Ca<sup>2+</sup> and 10 μM cis (cytoplasmic) Ca<sup>2+</sup>. (F–H) Average open probability (F), mean open time (G), and mean closed time (H) for 30-s recordings from four WT and four ΔM<sub>1,2,3</sub> channels in the presence of 10 μM cis Ca<sup>2+</sup> and combined data for 1 and 5 mM cis Ca<sup>2+</sup>. *, P < 0.05. (I) Data from a ΔM<sub>1,2,3</sub> channel recorded first in 100 nM cis Ca<sup>2+</sup> (top) and then in 10 μM cis Ca<sup>2+</sup> (bottom). The open probability for 30 s of channel activity at each Ca<sup>2+</sup> concentration is given above each record. (J) Average single channel conductance from four WT RyR1 channels and four ΔM<sub>1,2,3</sub> RyR1 channels.</p></caption><graphic xlink:href="jgp1300365f05"></graphic></fig><p>We next compared the function of purified WT and ΔM<sub>1,2,3</sub> channels following expression in HEK293 cells (<xref rid="fig5" ref-type="fig">Fig. 5</xref>). WT and ΔM<sub>1,2,3</sub> channels exhibited identical Ca<sup>2+</sup> dependence of [<sup>3</sup>H]ryanodine binding, with binding (which indirectly reflects P<sub>o</sub>) increasing with channel activation between 100 nM and 10 μM cis Ca<sup>2+</sup> (EC<sub>50</sub> was 4.14 ± 0.34 and 3.99 ± 0.17 μM for WT and ΔM<sub>1,2,3</sub> channels, respectively) and then similarly declining as cis Ca<sup>2+</sup> rises above 10 μM (<xref rid="fig5" ref-type="fig">Fig. 5 A</xref>). The data in <xref rid="fig5" ref-type="fig">Fig. 5 A</xref> are expressed relative to maximum binding in 10 μM Ca<sup>2+</sup> in order to reduce variations between ER preparations. Maximal [<sup>3</sup>H]ryanodine binding was not significantly different between WT and ΔM<sub>1,2,3</sub> channels (B<sub>max</sub> was 4.8 ± 0.6 and 4.5 ± 0.1 pM/mg for WT and ΔM<sub>1,2,3</sub> channels, respectively). Preincubation of recombinant WT RyR1 channels with 5 μg/ml triadin increased [<sup>3</sup>H]ryanodine binding relative to binding in the absence of triadin. In contrast, incubation with triadin had no effect on maximal [<sup>3</sup>H]ryanodine binding to ΔM<sub>1,2,3</sub> RyR1 (<xref rid="fig5" ref-type="fig">Fig. 5 B</xref>). These results demonstrate a strong functional consequence of triadin binding to WT RyR1 and the lack of any functional change when triadin is added to triadin binding-deficient ΔM<sub>1,2,3</sub> channels. Previous studies have shown an inhibitory interaction between the cytoplasmic domain of triadin and RyR1 (<xref ref-type="bibr" rid="bib28">Ohkura et al., 1998</xref>; <xref ref-type="bibr" rid="bib11">Groh et al., 1999</xref>).</p><p>The activity of single ΔM<sub>1,2,3</sub> channels in bilayers was similar to that of WT channels (<xref rid="fig5" ref-type="fig">Fig. 5, C–J</xref>). In six of eight experiments using either WT or ΔM<sub>1,2,3</sub> channels, current recordings showed periods where more than one channel opened in the bilayer with similar openings in WT and mutant channels (<xref rid="fig5" ref-type="fig">Fig. 5 C</xref>). To include data from these recordings, open probability of 30-s recordings was calculated as the mean current in the recording (average of all data points) normalized to the maximum current (maximum open level), i.e., I<sub>mean</sub>/I<sub>max</sub> (<xref rid="fig5" ref-type="fig">Fig. 5 D</xref>) (<xref ref-type="bibr" rid="bib3">Beard et al., 2002</xref>). In each experiment, channel activity was initially recorded in 5 mM cis Ca<sup>2+</sup> to aid in channel incorporation, cis Ca<sup>2+</sup> concentration was then decreased to 10 μM and subsequently returned to 1 mM. Since there was no significant difference in channel activity between 1 or 5 mM cis Ca<sup>2+</sup>, data at these two concentrations were combined and reported as ≥1 mM in <xref rid="fig5" ref-type="fig">Fig. 5</xref> (D–H). In agreement with the [<sup>3</sup>H]ryanodine binding data (<xref rid="fig5" ref-type="fig">Fig. 5 A</xref>) and previous studies (<xref ref-type="bibr" rid="bib23">Laver et al., 1997</xref>), WT channel activity fell significantly when the cis Ca<sup>2+</sup> concentration was increased from 10 μM to ≥1 mM Ca<sup>2+</sup>. However, no significant difference was observed between WT and ΔM<sub>1,2,3</sub> channels (<xref rid="fig5" ref-type="fig">Fig. 5, D and F</xref>). All recordings showed periods (10–30 s) of single channel opening (<xref rid="fig5" ref-type="fig">Fig. 5 C</xref>) that were also analyzed for open probability, mean open time, and mean closed time using a standard 20% threshold-crossing criterion (see Materials and methods). Open probability values obtained with this method (<xref rid="fig5" ref-type="fig">Fig. 5 F</xref>) were very similar to those estimated from I<sub>mean</sub>/I<sub>max</sub> (<xref rid="fig5" ref-type="fig">Fig. 5 D</xref>). The decline in open probability when cis Ca<sup>2+</sup> concentration was increased from 10 μM to ≥1 mM was due to an abbreviation of the mean open time (<xref rid="fig5" ref-type="fig">Fig. 5 G</xref>) and prolongation of the mean closed time (<xref rid="fig5" ref-type="fig">Fig. 5 H</xref>). These changes in mean open and closed times were similar in WT and ΔM<sub>1,2,3</sub> channels.</p><p>In one experiment with a ΔM<sub>1,2,3</sub> channel, the cis Ca<sup>2+</sup> concentration was lowered to 100 nM and then increased to 10 μM (<xref rid="fig5" ref-type="fig">Fig. 5 I</xref>). As expected from the [<sup>3</sup>H]ryanodine binding data (<xref rid="fig5" ref-type="fig">Fig. 5 A</xref>), the open probability of this channel was greater with 10 μM cis Ca<sup>2+</sup> than with 100 nM cis Ca<sup>2+</sup>, increasing from 0.15 to 0.66; the mean open time increased from 10.9 to 54.4 ms, and the mean closed time fell from 58.3 to 27.4 ms. The changes in open probability, mean open time, and mean closed time when Ca<sup>2+</sup> concentration was increased from 100 nM to 10 μM in this ΔM<sub>1,2,3</sub> channel are similar to changes in the activity of WT channels that occur over the same range of Ca<sup>2+</sup> concentrations. Finally, the ΔM<sub>1,2,3</sub> mutation did not significantly affect unitary conductance of RyR1 channels (<xref rid="fig5" ref-type="fig">Fig. 5 J</xref>) and the conductance at +40 mV was similar to that at −40 mV (<xref rid="fig5" ref-type="fig">Fig. 5</xref>, compare C with E or I). Average Cs<sup>+</sup> conductance was 222 ± 19 pS for WT RyR1 and 207 ± 24 pS for ΔM<sub>1,2,3</sub>. Taken together, the data in <xref rid="fig4" ref-type="fig">Figs. 4</xref> and <xref rid="fig5" ref-type="fig">5</xref> demonstrate that WT and ΔM<sub>1,2,3</sub> channels exhibit similar channel function in the absence of triadin.</p></sec><sec><title>Effects of Other RyR1 Luminal Loop Mutations on Ligand- and Voltage-gated Ca<sup>2+</sup> Release</title><p>Indo-1 Ca<sup>2+</sup> measurements in intact myotubes (<xref rid="fig6" ref-type="fig">Fig. 6</xref>) and whole-cell voltage clamp (<xref rid="fig7" ref-type="fig">Fig. 7</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>) experiments were also conducted in myotubes expressing each of the single (ΔM<sub>1</sub>, ΔM<sub>2</sub>, and ΔM<sub>3</sub>) and double (ΔM<sub>1,2</sub>, ΔM<sub>2,3</sub>, and ΔM<sub>1,3</sub>) RyR1 mutations. Electrically evoked release (<xref rid="fig6" ref-type="fig">Fig. 6 A</xref>), ligand-induced release (<xref rid="fig6" ref-type="fig">Fig. 6, B–D</xref>), and both retrograde (<xref rid="fig7" ref-type="fig">Fig. 7 A</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>) and orthograde coupling (<xref rid="fig7" ref-type="fig">Fig. 7 B</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>) were not significantly different between WT RyR1-expressing myotubes and either ΔM<sub>1</sub>-, ΔM<sub>2</sub>-, ΔM<sub>3</sub>-, or ΔM<sub>1,3</sub>-expressing myotubes. On the other hand, effects of the ΔM<sub>2,3</sub> mutation were similar to those of ΔM<sub>1,2,3</sub> in that ΔM<sub>2,3</sub>- expressing myotubes also lacked electrically evoked Ca<sup>2+</sup> release (<xref rid="fig6" ref-type="fig">Fig. 6 A</xref>), exhibited slowed kinetics of caffeine-induced Ca<sup>2+</sup> release (<xref rid="fig6" ref-type="fig">Fig. 6, C and D</xref>), normal L-type Ca<sup>2+</sup> channel activity (<xref rid="fig7" ref-type="fig">Fig. 7 A</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>), and lacked sigmoidal voltage-gated Ca<sup>2+</sup> release (<xref rid="fig7" ref-type="fig">Fig. 7 B</xref>). Interestingly, reduced triadin binding to the ΔM<sub>1,2</sub> mutant (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>) was accompanied by a significant reduction in the magnitude of electrically evoked Ca<sup>2+</sup> release (<xref rid="fig6" ref-type="fig">Fig. 6 A</xref>) and a slowing in ligand-induced Ca<sup>2+</sup> release (<xref rid="fig6" ref-type="fig">Fig. 6, C and D</xref>). In patch clamp experiments, there was a tendency (P = 0.05) for a reduction in voltage-gated Ca<sup>2+</sup> release measured at the end of 200-ms depolarizations in ΔM<sub>1,2</sub>-expressing myotubes (<xref ref-type="table" rid="tbl1">Table I</xref>). However, for release assessed 30 ms after the start of the test pulse (to approximate release during a brief action potential), voltage-gated Ca<sup>2+</sup> release (<xref rid="fig7" ref-type="fig">Fig. 7 C</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>) and the maximum rate of Ca<sup>2+</sup> release (<xref rid="fig7" ref-type="fig">Fig. 7 D</xref>), approximated from the peak of the first derivative of the Ca<sup>2+</sup> transient elicited at +70 mV (δ(ΔF/F)/δt), were significantly (P < 0.01) reduced in ΔM<sub>1,2</sub>-expressing myotubes.</p><fig position="float" id="fig6"><label>Figure 6.</label><caption><p>Effects of the terminal luminal loop RyR1 mutants on electrically evoked and caffeine-induced Ca<sup>2+</sup> release. (A–D) Bar graphs summarizing the effects of each of WT RyR1 and the different terminal RyR1 luminal loop mutants (ΔM<sub>1</sub>, ΔM<sub>2</sub>, ΔM<sub>3</sub>, ΔM<sub>1,2</sub>, ΔM<sub>1,3</sub>, ΔM<sub>2,3</sub>, and ΔM<sub>1,2,3</sub>) on electrically evoked Ca<sup>2+</sup> release (A), peak caffeine-induced (30 mM) Ca<sup>2+</sup> release (B), and the average time to peak (C) and <italic>t</italic><sub>1/2</sub> of decay (D) of caffeine-induced Ca<sup>2+</sup> transients.</p></caption><graphic xlink:href="jgp1300365f06"></graphic></fig><fig position="float" id="fig7"><label>Figure 7.</label><caption><p>Effects of the terminal luminal loop RyR1 mutants on orthograde and retrograde coupling. (A and B) Average voltage dependence of L-type Ca<sup>2+</sup> current density (A) and depolarization-induced Ca<sup>2+</sup> transients (B) in ΔM<sub>1</sub>-, ΔM<sub>2</sub>-, ΔM<sub>3</sub>-, ΔM<sub>1,2</sub>-, ΔM<sub>1,3</sub>-, and ΔM<sub>2,3</sub>-expressing myotubes. Dashed lines representing the average voltage dependence obtained from WT-expressing myotubes are shown for comparison. (C) Representative depolarization-induced (test potential = +70 mV) Ca<sup>2+</sup> transients from WT RyR1- and ΔM<sub>1,2</sub>-expressing myotubes (top). Voltage dependence of Ca<sup>2+</sup> transients measured 30 ms after the start of the test pulse (bottom). (D) Differentials of fluorescence traces (+70 mV) taken during the initial phase of depolarization (top). Bar graph of average peak differential (δ(ΔF/F)/δt) (bottom). The number of experiments is given within each bar. *, P < 0.01.</p></caption><graphic xlink:href="jgp1300365f07"></graphic></fig><table-wrap position="float" id="tbl1"><label>TABLE I</label><caption><p>Parameters of Fitted IV and FV Curves</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="top"></th><th colspan="1" rowspan="1" align="center"><italic>G<sub>max</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>k<sub>G</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>V<sub>G1/2</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>V<sub>rev</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>(</italic>Δ<italic>F/F)<sub>max</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>k<sub>F</sub></italic></th><th colspan="1" rowspan="1" align="center"><italic>V<sub>F1/2</sub></italic></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center"><italic>nS/nF</italic></td><td colspan="1" rowspan="1" align="center"><italic>mV</italic></td><td colspan="1" rowspan="1" align="center"><italic>mV</italic></td><td colspan="1" rowspan="1" align="center"><italic>mV</italic></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center"><italic>mV</italic></td><td colspan="1" rowspan="1" align="center"><italic>mV</italic></td></tr><tr><td colspan="1" rowspan="1" align="left">RyR1 (<italic>n</italic> = 27)</td><td colspan="1" rowspan="1" align="center">246 ± 12</td><td colspan="1" rowspan="1" align="center">4.8 ± 0.2</td><td colspan="1" rowspan="1" align="center">9.8 ± 1.0</td><td colspan="1" rowspan="1" align="center">74.6 ± 1.5</td><td colspan="1" rowspan="1" align="center">2.5 ± 0.2</td><td colspan="1" rowspan="1" align="center">4.8 ± 0.3</td><td colspan="1" rowspan="1" align="center">−2.7 ± 0.9</td></tr><tr><td colspan="1" rowspan="1" align="left">RyR1 (<italic>n</italic> = 27) (30 ms)</td><td colspan="1" rowspan="1" align="center">175 ± 10</td><td colspan="1" rowspan="1" align="center">5.5 ± 0.2</td><td colspan="1" rowspan="1" align="center">12.0 ± 1.0</td><td colspan="1" rowspan="1" align="center">70.9 ± 1.3</td><td colspan="1" rowspan="1" align="center">1.4 ± 0.1</td><td colspan="1" rowspan="1" align="center">5.1 ± 0.3</td><td colspan="1" rowspan="1" align="center">2.7 ± 0.9</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>1</sub> (<italic>n</italic> = 17)</td><td colspan="1" rowspan="1" align="center">232 ± 9</td><td colspan="1" rowspan="1" align="center">4.7 ± 0.5</td><td colspan="1" rowspan="1" align="center">8.7 ± 1.4</td><td colspan="1" rowspan="1" align="center">74.5 ± 1.7</td><td colspan="1" rowspan="1" align="center">2.1 ± 0.2</td><td colspan="1" rowspan="1" align="center">3.6 ± 0.5</td><td colspan="1" rowspan="1" align="center">−5.1 ± 0.9</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>2</sub> (<italic>n</italic> = 18)</td><td colspan="1" rowspan="1" align="center">247 ± 14</td><td colspan="1" rowspan="1" align="center">5.0 ± 0.4</td><td colspan="1" rowspan="1" align="center">6.5 ± 1.5</td><td colspan="1" rowspan="1" align="center">75.7 ± 2.1</td><td colspan="1" rowspan="1" align="center">2.3 ± 0.3</td><td colspan="1" rowspan="1" align="center">4.2 ± 0.5</td><td colspan="1" rowspan="1" align="center">−2.0 ± 1.8</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>3</sub> (<italic>n</italic> = 12)</td><td colspan="1" rowspan="1" align="center">211 ± 17</td><td colspan="1" rowspan="1" align="center">4.6 ± 0.3</td><td colspan="1" rowspan="1" align="center">11.1 ± 0.9</td><td colspan="1" rowspan="1" align="center">68.6 ± 1.8</td><td colspan="1" rowspan="1" align="center">2.0 ± 0.2</td><td colspan="1" rowspan="1" align="center">3.6 ± 0.3</td><td colspan="1" rowspan="1" align="center">−2.4 ± 0.7</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>1,2</sub> (<italic>n</italic> = 12)</td><td colspan="1" rowspan="1" align="center">180 ± 11</td><td colspan="1" rowspan="1" align="center">4.5 ± 0.3</td><td colspan="1" rowspan="1" align="center">9.2 ± 1.4</td><td colspan="1" rowspan="1" align="center">76.6 ± 2.3</td><td colspan="1" rowspan="1" align="center">1.8 ± 0.3</td><td colspan="1" rowspan="1" align="center">2.9 ± 0.3</td><td colspan="1" rowspan="1" align="center">1.9 ± 1.1</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>1,2</sub> (<italic>n</italic> = 12) (30 ms)</td><td colspan="1" rowspan="1" align="center">132 ± 10</td><td colspan="1" rowspan="1" align="center">5.7 ± 0.2</td><td colspan="1" rowspan="1" align="center">12.3 ± 1.8</td><td colspan="1" rowspan="1" align="center">70.3 ± 2.0</td><td colspan="1" rowspan="1" align="center">0.8 ± 0.1<xref ref-type="table-fn" rid="tblfn2">b</xref></td><td colspan="1" rowspan="1" align="center">4.8 ± 0.3</td><td colspan="1" rowspan="1" align="center">7.0 ± 1.1<xref ref-type="table-fn" rid="tblfn2">b</xref></td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>1,3</sub>(<italic>n</italic> = 6)</td><td colspan="1" rowspan="1" align="center">259 ± 14</td><td colspan="1" rowspan="1" align="center">4.8 ± 0.3</td><td colspan="1" rowspan="1" align="center">8.9 ± 1.0</td><td colspan="1" rowspan="1" align="center">73.6 ± 2.3</td><td colspan="1" rowspan="1" align="center">2.4 ± 0.3</td><td colspan="1" rowspan="1" align="center">3.7 ± 0.6</td><td colspan="1" rowspan="1" align="center">−5.0 ± 2.5</td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>2,3</sub> (<italic>n</italic> = 17)</td><td colspan="1" rowspan="1" align="center">187 ± 8</td><td colspan="1" rowspan="1" align="center">4.2 ± 0.4</td><td colspan="1" rowspan="1" align="center">6.9 ± 1.2</td><td colspan="1" rowspan="1" align="center">75.5 ± 2.1</td><td colspan="1" rowspan="1" align="center">0.9 ± 0.1<xref ref-type="table-fn" rid="tblfn1">a</xref></td><td colspan="1" rowspan="1" align="center">3.4 ± 0.3</td><td colspan="1" rowspan="1" align="center">10.5 ± 1.3<xref ref-type="table-fn" rid="tblfn1">a</xref></td></tr><tr><td colspan="1" rowspan="1" align="left">ΔM<sub>1,2,3</sub> (<italic>n</italic> = 18)</td><td colspan="1" rowspan="1" align="center">200 ± 11</td><td colspan="1" rowspan="1" align="center">3.6 ± 0.2</td><td colspan="1" rowspan="1" align="center">13.5 ± 1.2</td><td colspan="1" rowspan="1" align="center">75.0 ± 2.0</td><td colspan="1" rowspan="1" align="center">0.8 ± 0.1<xref ref-type="table-fn" rid="tblfn1">a</xref></td><td colspan="1" rowspan="1" align="center">4.0 ± 0.3</td><td colspan="1" rowspan="1" align="center">4.3 ± 1.1<xref ref-type="table-fn" rid="tblfn1">a</xref></td></tr></tbody></table><table-wrap-foot><fn><p>Values represent mean ± SEM for <italic>n</italic> experiments. Parameters for the voltage dependence of Ca<sup>2+</sup> current (<italic>I-V</italic>) and Ca<sup>2+</sup> release (Δ<italic>F</italic>/<italic>F</italic>) were obtained by fitting myotubes within each group separately to the appropriate equation (<italic>I-V</italic>, <xref ref-type="disp-formula" rid="fd1">Eq. 1</xref>; Δ<italic>F</italic>/<italic>F</italic>, <xref ref-type="disp-formula" rid="fd2">Eqs. 2</xref> and <xref ref-type="disp-formula" rid="fd3">3</xref>) as described in Materials and methods. <italic>G<sub>max</sub></italic>, maximal L-channel conductance; (Δ<italic>F</italic>/<italic>F</italic>)<sub><italic>max</italic></sub>, maximal change in relative fluo-4 fluorescence; <italic>V<sub>rev</sub></italic>, L-channel reversal potential; <italic>V<sub>G</sub></italic><sub>1/2</sub> and <italic>V<sub>F</sub></italic><sub>1/2</sub>, potential at which G and F are half-maximal, respectively; <italic>k<sub>G</sub></italic> and <italic>k<sub>F</sub></italic>, slope factors for <italic>I-V</italic> and Δ<italic>F</italic>/<italic>F</italic>, respectively.</p></fn><fn id="tblfn1"><label>a</label><p>P < 0.01, compared to WT (200 ms).</p></fn><fn id="tblfn2"><label>b</label><p>P < 0.01, compared to WT (30 ms).</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Triadin Binding-deficient RyR1 Mutants Exhibit Normal Junctional Targeting</title><p>The reduction in voltage-gated Ca<sup>2+</sup> release documented in ΔM<sub>1,2</sub>-, ΔM<sub>2,3</sub>-, and ΔM<sub>1,2,3</sub>- expressing myotubes (<xref rid="fig7" ref-type="fig">Fig. 7, B and C</xref>) could result from a lack of proper targeting of the mutant release channels to DHPR-containing SR–sarcolemmal junctions. However, our finding that all three of these triadin binding-deficient mutant RyR1 channels restored retrograde DHPR Ca<sup>2+</sup> channel conductance to a similar degree as WT RyR1 (<xref rid="fig6" ref-type="fig">Fig. 6 A</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>) provides strong functional evidence that each mutant was expressed, properly targeted to SR–sarcolemmal junctions, and interacted with DHPRs present in the junction. Nevertheless, we also compared DHPR and RyR subcellular localization in WT-, ΔM<sub>1,2</sub>-, ΔM<sub>2,3</sub>-, and ΔM<sub>1,2,3</sub>-expressing myotubes in coimmunofluorescence labeling experiments (<xref rid="fig8" ref-type="fig">Fig. 8</xref>). Consistent with the observed restoration of retrograde coupling for each of these constructs, the RyR1 mutants that displayed defects in triadin binding and electrically evoked Ca<sup>2+</sup> release exhibited a punctate appearance (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, left) that overlapped with discrete clusters of DHPR fluorescence (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, middle). This similar punctate appearance indicates correct junctional targeting of each construct and is particularly evident in the merged images (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, right), in which green and red indicate RyR1 and DHPR, respectively, and yellow represents common regions of punctate appearance.</p><fig position="float" id="fig8"><label>Figure 8.</label><caption><p>Triadin binding-deficient RyR1 mutants exhibit normal targeting to DHPR-containing junctions. (A) Double immunofluorescence labeling in representative WT- (first row), ΔM<sub>1,2</sub>- (second row), ΔM<sub>2,3</sub>- (third row), and ΔM<sub>1,2,3</sub>-expressing (fourth row) myotubes with antibodies against RyR1 (left) and the DHPR (middle). The “merged images” (right) emphasize common regions of puncta (yellow foci) containing expressed RyR1 proteins and endogenous DHPRs in clusters that represent junctions of the SR with the sarcolemma.</p></caption><graphic xlink:href="jgp1300365f08"></graphic></fig></sec></sec><sec><title>DISCUSSION</title><p>Since its initial discovery in 1990 (<xref ref-type="bibr" rid="bib6">Brandt et al., 1990</xref>), triadin has been shown to be enriched and embedded in the SR membrane of the triad (<xref ref-type="bibr" rid="bib21">Knudson et al., 1993a</xref>), to interact directly with both CSQ and RyR1 (<xref ref-type="bibr" rid="bib12">Guo and Campbell, 1995</xref>), to inhibit RyR channels when applied to the cytoplasmic side (<xref ref-type="bibr" rid="bib28">Ohkura et al., 1998</xref>), to activate channels when applied to the luminal side (<xref ref-type="bibr" rid="bib13">Gyorke et al., 2004</xref>), and to contribute to the luminal Ca<sup>2+</sup> transduction machinery that regulates Ca<sup>2+</sup> release channel activity in skeletal muscle (<xref ref-type="bibr" rid="bib4">Beard et al., 2004</xref>). <xref ref-type="bibr" rid="bib25">Lee et al. (2004)</xref> identified three negatively charged amino acids (D4878, D4907, and E4908) in an RyR1 luminal loop that were each required for the isolated loop to bind a specific positively charged triadin KEKE motif (residues 200–232) (<xref rid="fig1" ref-type="fig">Fig. 1 A</xref>). Our results provide the following important and novel contributions with regard to triadin binding/regulation of RyR1: (a) a single mutation to any of these three negatively charged residues is not sufficient to modify RyR1–triadin association and the combination of the different mutations demonstrates that each residue contributes unequally (D4907 > E4908 > D4878) to triadin binding (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>), (b) the RyR1 luminal loop mutations do not directly alter release channel function (<xref rid="fig4" ref-type="fig">Figs. 4</xref> and <xref rid="fig5" ref-type="fig">5</xref>), (c) luminal loop mutations that disrupt triadin binding do not alter either junctin binding (<xref rid="fig1" ref-type="fig">Fig. 1 C</xref>) or RyR1 junctional targeting (<xref rid="fig8" ref-type="fig">Fig. 8</xref>), and (d) triadin binding to RyR1 ensures rapid and robust Ca<sup>2+</sup> release during both voltage- and ligand-induced activation (<xref rid="fig2" ref-type="fig">Figs. 2</xref>, <xref rid="fig3" ref-type="fig">3</xref>, <xref rid="fig6" ref-type="fig">6</xref>, and <xref rid="fig7" ref-type="fig">7</xref>; and <xref ref-type="table" rid="tbl1">Table I</xref>).</p><p>Recently, <xref ref-type="bibr" rid="bib24">Lee et al. (2006)</xref> found that triadin binding was reduced and the kinetics of caffeine-induced calcium release slowed following infection of RyR-null 1B5 myotubes with herpes simplex virus-1 virions packaged with either ΔM<sub>2</sub> or ΔM<sub>1,2,3</sub> compared with that observed for WT RyR1 (<xref ref-type="bibr" rid="bib24">Lee et al., 2006</xref>). We found a similar slowing in the kinetics of caffeine-induced calcium release in ΔM<sub>1,2,3</sub>-expressing primary dyspedic myotubes (<xref rid="fig2" ref-type="fig">Fig. 2 C</xref>). However, unlike <xref ref-type="bibr" rid="bib24">Lee et al. (2006)</xref>, we found that triadin binding to RyR1 as well as the kinetics of caffeine-induced calcium release were unaltered by the ΔM<sub>2</sub> mutation (<xref rid="fig6" ref-type="fig">Fig. 6, C and D</xref>). Differences between the two studies observed for the effects of the ΔM<sub>2</sub> mutation are not entirely clear, but may involve differences between the cells, expression methods, and biochemical/analytical approaches used in the two studies.</p><p>Importantly, results presented here are the first to characterize the impact of triadin binding to RyR1 on the orthograde and retrograde signals of EC coupling. Specifically, we found that triadin binding to RyR1 enhances release channel activity during both voltage and ligand activation and that this critical regulation of release channel activity ensures robust and rapid calcium release during skeletal muscle EC coupling. This idea is further supported by our observation that [<sup>3</sup>H]ryanodine binding to WT RyR1, but not ΔM<sub>1,2,3</sub>, is significantly enhanced by the addition of purified triadin (<xref rid="fig5" ref-type="fig">Fig. 5 B</xref>) and that triadin increases the open probability of RyR channels incorporated into planar lipid bilayers (<xref ref-type="bibr" rid="bib13">Gyorke et al., 2004</xref>). In addition, our study is also the first to demonstrate that the mutations in RyR1 used to disrupt triadin binding do not directly affect release channel activity, retrograde RyR1–DHPR signaling, or the ability of junctin to bind to RyR1.</p><p>Defects observed for the RyR1 mutants could result either from effects of the mutations on triadin binding, direct effects on the channel, or both. We show that these mutations do not directly affect RyR1 channel function; WT and ΔM<sub>1,2,3</sub> channels expressed in HEK293 cells exhibited similar Ca<sup>2+</sup> dependence of [<sup>3</sup>H]ryanodine binding (<xref rid="fig5" ref-type="fig">Fig. 5 A</xref>), open probability (<xref rid="fig5" ref-type="fig">Fig. 5, D and F</xref>), mean open (<xref rid="fig5" ref-type="fig">Fig. 5 G</xref>) and closed (<xref rid="fig5" ref-type="fig">Fig. 5 H</xref>) times, conductance (<xref rid="fig5" ref-type="fig">Fig. 5 J</xref>), and Ca<sup>2+</sup> release activity (<xref rid="fig4" ref-type="fig">Fig. 4, B–D</xref>). Moreover, purified triadin increased [<sup>3</sup>H]ryanodine binding to WT RyR1, but not to ΔM<sub>1,2,3</sub> (<xref rid="fig5" ref-type="fig">Fig. 5 B</xref>). In addition, WT RyR1 and triadin binding-deficient mutants expressed in dyspedic myotubes were activated by Ca<sup>2+</sup> (<xref rid="fig3" ref-type="fig">Fig. 3 D</xref>), caffeine (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>), 4-cmc (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>), and fully restored retrograde DHPR coupling (<xref rid="fig7" ref-type="fig">Fig. 7 A</xref> and <xref ref-type="table" rid="tbl1">Table I</xref>). The slight reduction in maximal caffeine-induced Ca<sup>2+</sup> release (<xref rid="fig6" ref-type="fig">Fig. 6 B</xref>) observed for the triadin binding-deficient mutants may result from a combination of slow Ca<sup>2+</sup> release inactivating some channels and the fact that triadin binding to RyR1 enhances Ca<sup>2+</sup> gating (<xref rid="fig5" ref-type="fig">Fig. 5 B</xref><italic>)</italic>. Finally, effects of the luminal loop mutants on voltage-gated Ca<sup>2+</sup> release coincided perfectly with independent biochemical determination of triadin binding; mutations that did not affect binding (ΔM<sub>1</sub>, ΔM<sub>2</sub>, ΔM<sub>3</sub>, and ΔM<sub>1,3</sub>) did not affect orthograde coupling, mutations that eliminated binding abolished orthograde coupling (ΔM<sub>1,2,3</sub> and ΔM<sub>2,3</sub>), and mutations that partially disrupted binding partially reduced orthograde coupling (ΔM<sub>1,2</sub>). Since the binding of purified triadin to purified WT or mutant RyR1 proteins in our experiments is likely to be thermodynamically different from in vivo binding that occurs in the SR, we cannot completely rule out the possibility that the effects of the RyR1 mutations on release and triadin binding are coincidentally related. Nevertheless, our results provide strong evidence that altered voltage- and ligand-induced Ca<sup>2+</sup> release of the ΔM<sub>1,2,3</sub>, ΔM<sub>1,2</sub>, and ΔM<sub>2,3</sub> mutants result from defects in triadin binding rather than direct effects of the mutations on channel function.</p><p><xref rid="fig9" ref-type="fig">Fig. 9</xref> provides a simple model that ties together previously published results with the findings of this study. Under normal conditions (<xref rid="fig9" ref-type="fig">Fig. 9</xref>, left), triadin binds to both CSQ and the terminal luminal loop of RyR1, forming a quaternary complex along with junctin that maximizes SR Ca<sup>2+</sup> release following activation of RyR1 by either endogenous (e.g., DHPR) or exogenous (e.g., caffeine or 4-cmc) triggers. In this model, mutations in RyR1 that interfere with triadin binding are proposed to disrupt this critical RyR1 regulatory mechanism and result in the dissociation of the triadin–CSQ complex from the release channel (<xref rid="fig9" ref-type="fig">Fig. 9</xref>, right). Since we found that junctin binding to RyR1 is not affected by any of the RyR1 luminal loop mutations, a junctin–CSQ complex is shown to interact with an alternate RyR1 site in <xref rid="fig9" ref-type="fig">Fig. 9</xref>, suggesting that RyR1 activity may be differentially regulated by triadin–CSQ and junctin–CSQ. Our results indicate that triadin binding to RyR1 primes the channel for ligand/voltage sensor activation in order to ensure rapid Ca<sup>2+</sup> release kinetics following either voltage- or ligand-induced activation. In the absence of this critical interaction of triadin–CSQ with RyR1, the kinetics of ligand- (<xref rid="fig2" ref-type="fig">Fig. 2 C</xref> and <xref rid="fig6" ref-type="fig">Fig. 6 C</xref>) and voltage-induced (<xref rid="fig7" ref-type="fig">Fig. 7, C and D</xref>) Ca<sup>2+</sup> release is markedly slowed. In the case of release stimulated by a brief action potential following neuromuscular transmission, this reduction in the rate of release is sufficient to essentially abolish Ca<sup>2+</sup> release during EC coupling (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref> and <xref rid="fig6" ref-type="fig">Fig. 6 A</xref>).</p><fig position="float" id="fig9"><label>Figure 9.</label><caption><p>Proposed model for triadin regulation of DHPR and ligand activation of RyR1. The quaternary CSQ–triadin–RyR1–junctin interaction tethers CSQ close to the release channel pore and promotes high probability release channel opening (depicted by a large arrow) following either DHPR or ligand activation (left). Disruption of triadin binding to RyR1 results in a similar reduction in SR Ca<sup>2+</sup> release (depicted by a small arrow) following either DHPR or ligand activation (right). A junctin–CSQ complex is shown to bind to a separate RyR1 site from that of triadin.</p></caption><graphic xlink:href="jgp1300365f09"></graphic></fig><p>Overexpression of triadin in rat skeletal myotubes was shown to significantly reduce KCl depolarization- induced Ca<sup>2+</sup> release with minimal effects on either caffeine-/4-cmc–induced Ca<sup>2+</sup> release or L-type Ca<sup>2+</sup> current density (<xref ref-type="bibr" rid="bib30">Rezgui et al., 2005</xref>). Triadin overexpression could result in either increased (due to enhanced levels of triadin) or decreased (due to overexpression causing triadin aggregation, <xref ref-type="bibr" rid="bib21">Knudson et al., 1993a</xref>) triadin binding to RyR1. In either case, the results of <xref ref-type="bibr" rid="bib30">Rezgui et al. (2005)</xref> indicate that the EC coupling machinery is exquisitely sensitive to triadin– RyR1 stoichiometry. Our results are consistent with this notion since disruption of triadin binding to RyR1 also markedly reduced orthograde, but not retrograde, DHPR–RyR1 coupling. The similar effects on voltage-gated Ca<sup>2+</sup> release observed following triadin overexpression and triadin dissociation from RyR1 could be explained by effects either of disrupting triadin binding to RyR1 or of free triadin on the EC coupling machinery.</p><p>Preliminary reports have produced conflicting results regarding effects of triadin deficiency on EC coupling. For example, KCl-induced Ca<sup>2+</sup> release is similar in myotubes derived from WT and pan-triadin knockout mice (Shen, X., J.R. Lopez, P.D. Allen, and C.F. Perez. 2006. Biophysical Society Meeting. Abstr. 328) but significantly reduced and slowed following transient siRNA-mediated knockdown of triadin and junctin in C2C12 myotubes (Wang, Y., L. Xinghai, H. Duan, T. Fulton, and G. Meissner. 2007. Biophysical Society Meeting. Abstr. 1237). The second finding is consistent with our results. The absence of an effect on depolarization-induced Ca<sup>2+</sup> release in pan-triadin knockout mice could result from a multitude of different compensatory mechanisms commandeered to correct for the loss of a critical RyR1 regulatory protein. In fact, skeletal muscle function and EC coupling is also not markedly altered following knockout of the skeletal muscle isoform of calsequestrin (CSQ1; <xref ref-type="bibr" rid="bib29">Paolini et al., 2007</xref>). Similarly, cardiac SR Ca<sup>2+</sup> storage and depolarization-induced Ca<sup>2+</sup> release are essentially normal following either global knockout of CSQ2 (<xref ref-type="bibr" rid="bib20">Knollmann et al., 2006</xref>) or cardiac-specific knockout of the Na/Ca exchanger (<xref ref-type="bibr" rid="bib14">Henderson et al., 2004</xref>), respectively. The surprising lack of an effect of genetic ablation of either CSQ1/2 or the Na/Ca exchanger on EC coupling certainly does not mean that these proteins are not normally important for calcium storage and removal, respectively. Rather, remarkable compensatory changes in Ca<sup>2+</sup> release unit assembly and efficiency were found to counteract CSQ and Na/Ca exchange deficiency in these animals. Conceivably, similar compensatory changes might also account for the absence of a marked effect of triadin ablation of skeletal muscle EC coupling.</p><p>More than 15 different central core disease mutations in RyR1 have been identified in the terminal RyR1 luminal loop shown here to markedly influence triadin binding and ligand/voltage-gated Ca<sup>2+</sup> release. A number of these mutations reduce Ca<sup>2+</sup> release without significantly affecting either SR Ca<sup>2+</sup> content or retrograde coupling to the DHPR (<xref ref-type="bibr" rid="bib2">Avila et al., 2003</xref>). Thus, it will be important for future studies to determine if any of these disease mutations in the RyR1 terminal luminal loop diminishes Ca<sup>2+</sup> release during EC coupling by altering the critical regulatory interaction of triadin with the SR Ca<sup>2+</sup> release channel.</p></sec></body><back><ack><p>We would like to thank Dr. Paul D. Allen (Brigham and Women's Hospital, Harvard Medical School, Boston, MA) for providing access to the dyspedic mice used in this study and to Linda Groom for excellent technical assistance. The anti-junctin antibody used in this study was generously provided by Dr. Steve Cala (Wayne State University, Detroit, MI).</p><p>This work was supported by research grants from the National Institutes of Health (AR44657 to R.T. Dirksen) and the Australian National Health and Medical Research Council (316937 to A.F. Dulhunty).</p><p>Olaf S. Andersen served as editor.</p></ack><ref-list><ref id="bib1"><citation citation-type="journal">Avila, G., J.J. O'Brien, and R.T. Dirksen. <year>2001</year>. Excitation-contraction uncoupling by a human central core disease mutation in the ryanodine receptor. <source>Proc. Natl. Acad. Sci. 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