MODULATION OF SKELETAL-MUSCLE CA2-RELEASE CHANNEL ACTIVITY BY SPHINGOSINE()

The effect of D-erythro-C-18-sphingosine (sphingosine) and related com pounds on the Ca2+-release channel (ryanodine binding protein) was exa mined on rabbit skeletal muscle membranes, on the purified ryanodine b inding protein, and on the channel reconstituted into planar lipid bil ayers. Sphingosine inhibited [H-3]ryanodine binding to sarcoplasmic re ticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, a nd G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolip id also inhibited [H-3]ryanodine binding to the purified ryanodine bin ding protein. Our results demonstrate that the inhibition of [H-3]ryan odine binding by sphingosine is due to an increased rate of dissociati on of bound [H-3]ryanodine from SR membranes and a decreased rate of a ssociation of [H-3]ryanodine to the high-affinity site. Unlike other m odulators of the Ca2+-release channel, sphingosine can remove bound [H -3]ryanodine from the high-affinity site within minutes. Sphingosine i ncreased the rate of dissociation of [H-3]ryanodine bound to a solubil ized proteolytic fragment derived from the carboxy terminus of the rya nodine binding protein (cleavage at Arg(4475)). Sphingosine also inhib ited the activity of the Ca2+-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direc t effect of sphingosine on the Ca2+-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg(4475) and the carboxy terminu s of the Ca2+-release channel.