Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.
Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.
Anderson
K
K
Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Lai
F A
FA
Liu
Q Y
QY
Rousseau
E
E
Erickson
H P
HP
Meissner
G
G
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004285:Dogs; D006352:Heart Ventricles / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009928:Organ Specificity; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D007473:Ion Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium