Chen S R, Ebisawa K, Li X, Zhang L
Cardiovascular Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 1998 Jun 12;273(24):14675-8. doi: 10.1074/jbc.273.24.14675.
We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR.
我们通过定点诱变以及功能检测（包括平面脂双层中的钙离子释放测量和单通道记录）研究了钙离子激活兰尼碱受体（RyR）的分子基础。我们在此报告，在3885位（位于3型RyR假定的跨膜序列M2中）将谷氨酸替换为丙氨酸的单个突变，通过单通道激活测量，可使钙离子敏感性降低超过10000倍，而通道电导以及其他配体（如ATP和兰尼碱）的调节作用无明显变化。野生型和突变型RyR蛋白的共表达导致合成具有中等钙离子敏感性的单通道。这些结果表明，每个单体3885位的谷氨酸可能以协同方式发挥作用，在对应于RyR的四聚体结构中形成钙离子传感器。
