Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel.
The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002846:Chromatography, Affinity; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000166:cytology*; D010539:Permeability; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000032:analysis*
D007473:Ion Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium