High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.
The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.
Rardon
D P
DP
Krannert Institute of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Cefali
D C
DC
Mitchell
R D
RD
Seiler
S M
SM
Jones
L R
LR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis* / Q000378:metabolism; D004285:Dogs; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis* / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification*; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000032:analysis* / Q000378:metabolism
D000470:Alkaloids; D015220:Calcium Channels; D011506:Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D000255:Adenosine Triphosphate; D002118:Calcium