LOCALIZATION OF THE HIGH AND LOW-AFFINITY [H-3] RYANODINE BINDING-SITES ON THE SKELETAL-MUSCLE CA2+ RELEASE CHANNEL

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant allialoid ryanodine and th ere are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino ac ids. The ability of sarcoplasmic reticulum membranes to bind [H-3]ryan odine to the high affinity site is lost upon proteolysis with trypsin. [H-3]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [ H-3]ryanodine and then 100 mu M ryanodine is added to occupy the low a ffinity sites, almost all of [H-3]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubili zed Ca2+ release channel containing bound [H-3]ryanodine to undergo fo ur discrete shifts in sedimentation (30 S --> 28 S --> 26 S --> 19 S - -> 14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56 -, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are rec ognized by an antibody to the last 9 amino acids at the carboxyl termi nus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37 -kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2release channel fragments. Both high and low affinity ryanodine bindin g sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.