PDF(Pubmed)
Abstract：
Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels\' pore cytosolic exit affects the channel\'s gating behavior thereby explaining the enhanced ligand-channel\'s sensitivity.
摘要：
肌醇1,4,5-三磷酸受体（IP3R）和ryanodine受体（RyR）是介导Ca2从内质网/肌浆网（ER/SR）释放的同源阳离子通道，从而参与许多生理过程。在以往的研究中,我们确定，当D2594残留物，位于IP3R1型的门处或附近，被赖氨酸(D2594K)取代，获得了功能的增益。该突变表型的特征在于增加的IP3敏感性。我们假设IP3R1-D2594通过静电影响封闭和开放状态的稳定性来确定通道的配体敏感性。为了测试这种可能性，D2594位点与IP3调节IP3R1之间的关系，并在细胞中测定了腔内的Ca2+，亚细胞，和单通道水平使用荧光Ca2+成像和单通道重建。我们发现在细胞中，D2594K突变增强IP3配体敏感性。单通道IP3R1研究表明，IP3R1-WT和-D2594K通道的电导相似。然而,IP3R1-D2594K通道具有更高的IP3灵敏度，具有更大的功效。此外,像它的野生型(WT)对应物一样，IP3R1-D2594K显示钟形细胞溶质Ca2+-依赖性，但D2594K在每个测试的胞浆游离Ca2+浓度下具有更大的活性。IP3R1-D2594K还具有改变的腔Ca2+敏感性。与IP3R1-WT不同，D2594K通道活性在低腔Ca2+水平下没有降低。一起来看,我们的功能研究表明，在通道孔胞浆出口处，带负电荷的残基被正电荷的残基取代会影响通道的门控行为，从而解释了配体通道敏感性的增强。
