肌醇1,4,5-三磷酸(IP3)受体由三种亚型组成:IP3R1,IPR2和IPR3。尽管已经合成了许多IP受体配体,没有亚型选择性配体是已知的。我们开发了一种简单的荧光方法,使用基于FRET的IP生物传感器LIBRAvI检查IP受体配体的亚型选择性,LIBRAvII,LIBRAVIII向透化的生物传感器表达细胞中添加IP或腺样体A(ADA)以浓度依赖的方式增加了这些生物传感器的荧光比率,根据LIBRAvI的荧光比的变化,ADA相对于IP的效力,LIBRAvII,LIBRAvIII是43-,22-,28倍,分别。这种基于荧光的方法进一步表明,几种ADA类似物在亚型选择性和效力方面具有显着差异。这些结果突出了ADA的O-糖苷结构在配体对IP3R1的选择性中所起的重要作用,这由5'-羟基被苯基或苯乙基取代后的选择性修饰所证明。我们还发现,一种ADA类似物5'-脱氧-5'-苯基腺苷酸A对IP3R1具有部分激动作用。一起,本文所述的新型荧光方法可用于评估IP3R配体的性质,包括效力,功效,和亚型选择性。
Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5\'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5\'-deoxy-5\'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity.