Biochemical characterization and molecular cloning of cardiac triadin.
Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling.
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
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W
Guo
A O
AO
Jorgensen
L R
LR
Jones
K P
KP
Campbell
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000378:metabolism; D002155:Calsequestrin / Q000378:metabolism; D002352:Carrier Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006321:Heart / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism*; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D015220:Calcium Channels; D002155:Calsequestrin; D002352:Carrier Proteins; D018076:DNA, Complementary; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013439:Sulfhydryl Reagents; C070048:triadin