Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum.
The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.
Smith
J S
JS
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Imagawa
T
T
Ma
J
J
Fill
M
M
Campbell
K P
KP
Coronado
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004553:Electric Conductivity; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D007477:Ions; D010539:Permeability; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012996:Solutions; D014325:Tromethamine / Q000494:pharmacology
D007473:Ion Channels; D007477:Ions; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012996:Solutions; D014325:Tromethamine; D012430:Ruthenium Red; D000255:Adenosine Triphosphate; D002118:Calcium