Du G G, Imredy J P, MacLennan D H
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1998 Dec 11;273(50):33259-66. doi: 10.1074/jbc.273.50.33259.
A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.
设计了一种用于检测高亲和力[³H]ryanodine与3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸(CHAPS)溶解的重组或天然Ca²⁺释放通道蛋白(ryanodine受体，RyR)结合的快速检测方法。在CHAPS浓度不断增加的情况下，保存高亲和力[³H]ryanodine结合位点的关键是添加磷脂酰胆碱。该检测方法用于表征[³H]ryanodine与重组骨骼肌(RyR1)和心脏(RyR2)Ca²⁺释放通道结合的平衡和动力学特性，以及包括ATP、Ca²⁺和Mg²⁺在内的生理调节剂对结合的影响。RyR1和RyR2都有一个单一的高亲和力ryanodine结合位点和低亲和力位点，但[³H]ryanodine与重组RyR2的结合对ATP激活或Ca²⁺失活不敏感，对Mg²⁺抑制也较不敏感。[³H]ryanodine结合检测用于估计重组RyR2和RyR1的表达水平，并表明RyR2可以在HEK-293细胞中以非常高的水平表达。通过测量细胞内Fura-2荧光来分析重组RyR2和RyR1的特性，结果表明RyR2和RyR1的不同特性在重组表达的蛋白中得以保留。
