Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor.
Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.
Groh
S
S
Laboratoire Canaux Ioniques et Signalisation, CEA/DBMS, 17 rue des Martyrs, 38054 Grenoble, France.
Marty
I
I
Ottolia
M
M
Prestipino
G
G
Chapel
A
A
Villaz
M
M
Ronjat
M
M
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002352:Carrier Proteins; D003593:Cytoplasm / Q000378:metabolism*; D009124:Muscle Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin