Location of ryanodine receptor binding site on skeletal muscle triadin.
The binding between intact triadin or expressed triadin peptides and the ryanodine receptor has been investigated using membrane overlay and affinity chromatography. Ryanodine receptor binds to triadin blotted onto nitrocellulose with a KD of 40 nM in a medium containing 150 mM NaCl. The binding is substantially inhibited by hypertonic salt solution. Blot overlay experiments show that ryanodine receptor binds to bacterially expressed peptides, triadin(110-280), triadin(110-267), and triadin(279-674), but to no other moieties of the protein (numbers in parentheses are the residue positions). This binding is strongly inhibited by hypertonic salt solution. The same three triadin peptides as well as triadin(68-267), when attached to a glutathione column, bind to the ryanodine receptor. However, triadin(110-280) binds with high affinity, while triadin(68-267), triadin(110-267), and triadin(279-674) bind with low affinity. Triadin(258-280), triadin(267-280), and triadin(258-299) all bind to the ryanodine receptor with high affinity. On the other hand, a construct containing triadin(267-280), but preceded by nine residues of heterologous amino acids, does not bind significantly. These observations indicate two types of binding between triadin and the ryanodine receptor: (1) a low-affinity ionic interaction of large portions of triadin; (2) a specific high-affinity binding of a short relatively hydrophobic segment. The binding of this segment is probably the physiologically important domain for attachment between triadin and the ryanodine receptor.
Caswell
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AH
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33136, USA.
Motoike
H K
HK
Fan
H
H
Brandt
N R
NR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism
D002352:Carrier Proteins; D009124:Muscle Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin