Olivares E, Arispe N, Rojas E
Laboratory of Cell Biology and Genetics, National Institute for Diabetes, Digestive Diseases and Kidney, National Institutes of Health, Bethesda, MD 20892.
Membr Biochem. 1993 Oct-Dec;10(4):221-35. doi: 10.3109/09687689309150270.
Microsomal sarcoplasmic reticulum (SR) fractions from lobster skeletal muscle were found to bind [3H]-ryanodine. [3H]-ryanodine binding was enhanced by AMP, Ca2+ and caffeine, and significantly diminished by ATP, Ba2+ and Sr2+. Furthermore, dantrolene and ruthenium red, two classical inhibitors of Ca2+ release from the SR, blocked [3H]-ryanodine binding. Similarly, tetracaine, known to block the charge movement associated with excitation-contraction coupling in vertebrate muscle, inhibited the binding of the alkaloid. Our lobster SR preparation exhibited a single high-affinity ryanodine binding site (Kd = 6.6 nM, Bmax = 10 pmol/mg protein). Since SDS-PAGE of the SR proteins revealed a major band c. 565 kDa which comigrated with the putative ryanodine receptor from both rat and chicken skeletal muscle, we concluded that lobster skeletal muscle is equipped with the 565 kDa ryanodine receptor. Finally, incorporation of the SR microsomal fraction from lobster into planar bilayer membranes revealed the presence of a ryanodine-sensitive Ca2+ channel activity (160 pS in symmetrical 200 mM CsCl solutions). We concluded that both the crustacean and vertebrate skeletal muscle ryanodine receptor share the relevant properties such as molecular weight and affinity for ryanodine and inositol 1,4,5 triphosphate. However, there are important differences between the two receptors including differential effects of the alkaloid on the Ca2+ release channel and modulation of the receptor by nucleotides.
研究发现，龙虾骨骼肌的微粒体肌浆网（SR）组分能结合[3H]-ryanodine。AMP、Ca2+和咖啡因可增强[3H]-ryanodine的结合，而ATP、Ba2+和Sr2+则使其显著减少。此外，丹曲林和钌红这两种经典的肌浆网Ca2+释放抑制剂可阻断[3H]-ryanodine的结合。同样，已知可阻断脊椎动物肌肉中与兴奋-收缩偶联相关电荷移动的丁卡因，也能抑制该生物碱的结合。我们制备的龙虾SR制剂表现出一个单一的高亲和力ryanodine结合位点（Kd = 6.6 nM，Bmax = 10 pmol/mg蛋白）。由于SR蛋白的SDS-PAGE显示出一条约565 kDa的主要条带，其迁移位置与大鼠和鸡骨骼肌中假定的ryanodine受体相同，因此我们得出结论，龙虾骨骼肌配备有565 kDa的ryanodine受体。最后，将龙虾的SR微粒体组分整合到平面双层膜中，发现存在对ryanodine敏感的Ca2+通道活性（在对称的200 mM CsCl溶液中为160 pS）。我们得出结论，甲壳类和脊椎动物骨骼肌的ryanodine受体具有一些相关特性，如分子量、对ryanodine和肌醇1,4,5-三磷酸的亲和力等。然而，这两种受体之间也存在重要差异，包括生物碱对Ca2+释放通道的不同影响以及核苷酸对受体的调节作用。
