METHODS: Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery.
RESULTS: mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1.
CONCLUSIONS: Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.
方法:正构激动剂,在表达五种突变mGlu1亚型的FlpInTrexHEK293A细胞系中,在两个功能终点(iCa2+动员和肌醇1-磷酸[IP1]积累)评估了正变构调节剂(PAMs)和负变构调节剂(NAMs)。关键药理学参数,包括配体效力,亲和力和协同性是使用激动和变形金刚的操作模型得出的。
结果:mGlu1突变体对mGlu1表达表现出不同的影响,C末端截短显著降低了表面表达。突变差异影响正位配体亲和力,变构和正构配体之间的功效和功能协同性。功能缺失突变L454F和N885del降低了正构亲和力和功效,分别。功能获得Y792C突变体mGlu1在IP1测定中显示出增强的组成活性,表现为正构激动剂活性降低。相对于野生型mGlu1,mGlu1PAM在iCa2动员中恢复了功能丧失突变的谷氨酸效力,并且mGlu1NAM在Y792C显示出增强的反向激动剂活性。
结论:总的来说,这些数据突出了mGlu1突变影响受体功能的不同机制,并显示变构调节剂可能为恢复罕见SCA亚型中mGlu1异常功能提供治疗策略.