Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin.
Normal Ca(2+) signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca(2+) binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca(2+) stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca(2+) fell from 1mM to <or=100 microM, as seen with native channels (containing endogenous calsequestrin/triadin/junctin). In contrast, skeletal calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca(2+) fell, as seen with purified channels prior to triadin/calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca(2+), skeletal calsequestrin and skeletal ryanodine receptors and may curtail resting Ca(2+) leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation-contraction coupling.
Wei
Lan
L
John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra City, ACT 2601, Australia.
Gallant
Esther M
EM
Dulhunty
Angela F
AF
Beard
Nicole A
NA
D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin; C498842:Junctin protein, Oryctolagus cuniculus; D006899:Mixed Function Oxygenases