Factors influencing [3H]ryanodine binding to the skeletal muscle Ca2+ release channel.
Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2+ concentration, ionic strength, and the presence of modulators of the Ca2+ release channel. The rate of association of [3H]-ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data.
Needleman
D H
DH
Department of Molecular Physiology and Biophysics, Boylor College of Medicine, Houston, Texas 77030, USA.
Hamilton
S L
SL
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002793:Cholic Acids / Q000378:metabolism*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D009994:Osmolar Concentration; D011817:Rabbits; D011869:Radioligand Assay; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D014316:Tritium
D015220:Calcium Channels; D002793:Cholic Acids; D003902:Detergents; D014316:Tritium; D012433:Ryanodine; D012710:Serum Albumin, Bovine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium