FACTORS INFLUENCING [H-3] RYANODINE BINDING TO THE SKELETAL-MUSCLE CA2+ RELEASE CHANNEL

Optimal [H-3]ryanodine binding to skeletal muscle sarcoplasmic reticul um membranes is dependent on a number of factors such as Ca2+ concentr ation, ionic strength, and the presence of modulators of the Ca2+ rele ase channel. The rate of association of [H-3]ryanodine with its bindin g site is slower than a diffusion limited process, and often the bindi ng reaches a peak value which is followed by a slow decline. This phen omenon makes it extremely difficult to determine kinetic constants for [H-3]ryanodine binding. The inclusion of bovine serum albumin (BSA) o r the detergent olamidopropyl)dimethylammonio]-1-propane-sulfonate (Ch aps) in the incubation buffer prevents the decrease in [H-3]ryanodine binding observed in association studies. BSA or Chaps slows this decli ne in binding partially by preventing a conversion to a more rapidly d issociating component. Pretreatment of the membranes with Chaps does n ot prevent the decrease in [H-3]ryanodine binding, suggesting that Cha ps is not exerting its effect by extracting a lipid or peripheral memb rane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryano dine binding site. Incubation for extended times in the absence of [H- 3]ryanodine prior to the initiation of the association produced simila r curves to those obtained without preincubation. These combined resul ts suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ re lease channel by preventing an alteration in the ryanodine binding sit e which leads to decreased affinity, thus allowing for a more quantita tive interpretation Of binding data. (C) 1997 Academic Press.