Wang J P, Needleman D H, Seryshev A B, Aghdasi B, Slavik K J, Liu S Q, Pedersen S E, Hamilton S L
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1996 Apr 5;271(14):8387-93. doi: 10.1074/jbc.271.14.8387.
Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.
新霉素是骨骼肌肌浆网（SR）钙释放的强效抑制剂。为阐明其抑制机制，研究了新霉素对[3H]ryanodine与Ca2+释放通道结合的影响以及重组到平面脂质双分子层时对其通道活性的影响。[3H]ryanodine的平衡结合受到新霉素的部分抑制。在高浓度新霉素下抑制不完全，表明是非竞争性抑制而非直接竞争性抑制。新霉素和[3H]ryanodine可同时结合到通道上，并且如果[3H]ryanodine先结合，加入新霉素会减慢[3H]ryanodine从高亲和力位点的解离。新霉素也会减慢[3H]ryanodine与高亲和力结合位点的结合。因此，新霉素结合位点似乎与ryanodine结合位点不同。新霉素也会减慢[3H]ryanodine从胰蛋白酶处理的膜或可溶性14S复合物上的解离。该复合物由Ca2+释放通道Arg-4475之后羧基末端衍生的多肽组成（卡拉韦，C.，谢里舍夫，A.，王，J.P.，斯拉维克，K.，尼德曼，D.H.，坎图，C.，吴，Y.，贾亚拉曼，T.，马克斯，A.R.，和汉密尔顿，S.L.（1994）《生物化学杂志》269，15876 - 15884）。与结合了ryanodine分离得到的蛋白水解14S复合物重组到平面脂质双分子层时会形成通道，其活性受到新霉素抑制。我们的数据与一种模型一致，即ryanodine结合位点、新霉素结合位点以及Ca2+释放通道的通道形成部分位于Arg-4475和羧基末端之间。
