Factors Influencing [3H]Ryanodine Binding to the Skeletal Muscle Ca2+Release Channel

Abstract

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-propane-sulfonate (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.