Tu Huiping, Nosyreva Elena, Miyakawa Tomoya, Wang Zhengnan, Mizushima Akiko, Iino Masamitsu, Bezprozvanny Ilya
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9040, USA.
Biophys J. 2003 Jul;85(1):290-9. doi: 10.1016/S0006-3495(03)74474-9.
Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.
胞质钙（Ca(2+)）对1型肌醇（1,4,5）-三磷酸受体（InsP(3)R1）的调节在其信号传导功能中起着至关重要的作用，但Ca(2+)对InsP(3)R1调节的结构决定因素和机制尚不清楚。在我们之前的研究中，利用DT40细胞表达系统和Ca(2+)成像分析，我们确定了E2100残基在Ca(2+)对InsP(3)R1调节中的关键作用。在本研究中，通过使用内源色氨酸荧光测量，我们确定假定的InsP(3)R1 Ca(2+)传感区域（E1932-R2270）以0.16微摩尔的亲和力结合Ca(2+)。我们进一步确定，E2100D和E2100Q突变将假定的InsP(3)R1 Ca(2+)传感区域的Ca(2+)结合亲和力降低到1微摩尔。在用草地贪夜蛾细胞中表达的重组InsP(3)R1进行的平面脂质双层实验中，我们发现E2100D和E2100Q突变将InsP(3)R1钟形Ca(2+)依赖性的峰值从0.2微摩尔转移到1.5微摩尔Ca(2+)。与生化数据一致，我们发现InsP(3)R1的Ca(2+)激活和抑制位点的表观亲和力对于野生型通道为0.2微摩尔，对于E2100D和E2100Q突变体为1-2微摩尔Ca(2+)。我们研究中获得的结果支持了E2100残基构成InsP(3)R1 Ca(2+)传感器一部分的假设。
