GABAB受体(GABABR)是异二聚体七跨膜受体,与一系列蛋白质相互作用,并在富含胆固醇的膜微结构域上形成大的蛋白质复合物。随着大脑的老化,膜胆固醇水平表现出变化,尽管目前尚不清楚这些变化如何影响蛋白-蛋白相互作用和下游信号。在这里,我们研究了GABABR和KCC2转运蛋白之间相互作用的结构基础,包括它们的蛋白质表达和分布,我们比较了年轻和老年大鼠小脑的数据。此外,我们分析了两组的血脂分布,我们对三种不同胆固醇浓度的质膜系统进行了分子动力学模拟,进一步探讨GABABR-转运体相互作用。根据我们的结果,我们报道了GABAB2亚基表达在老年大鼠小脑中显著下降.在进行比较免疫共沉淀分析后,我们证实GABABR和KCC2在成年和老年大鼠小脑中形成蛋白质复合物,尽管它们的相互作用水平随着小脑年龄的增长而大大降低。另一方面,我们的血脂分析显示,老年小脑的胆固醇和鞘磷脂水平显著升高。最后,我们使用Martini粗粒模型进行分子动力学模拟,从中我们观察到膜胆固醇浓度可以决定GABABR尾部结构域是否与转运蛋白物理建立不依赖G蛋白的接触,以及这些关联最终发生的时机。一起来看,我们的发现说明了与年龄相关的膜胆固醇水平变化如何影响蛋白质-蛋白质相互作用,以及它们如何在调节GABABR相互作用组介导的信号传导中发挥关键作用。
GABA B receptors (GABABRs) are heterodimeric seven-transmembrane receptors that interact with a range of proteins and form large protein complexes on cholesterol-rich membrane microdomains. As the brain ages, membrane cholesterol levels exhibit alterations, although it remains unclear how these changes impact protein-protein interactions and downstream signaling. Herein, we studied the structural bases for the interaction between GABABR and the
KCC2 transporter, including their protein expression and distribution, and we compared data between young and aged rat cerebella. Also, we analyzed lipid profiles for both groups, and we used molecular dynamics simulations on three plasma membrane systems with different cholesterol concentrations, to further explore the GABABR-transporter interaction. Based on our results, we report that a significant decrease in GABAB2 subunit expression occurs in the aged rat cerebella. After performing a comparative co-immunoprecipitation analysis, we confirm that GABABR and
KCC2 form a protein complex in adult and aged rat cerebella, although their interaction levels are reduced substantially as the cerebellum ages. On the other hand, our lipid analyses reveal a significant increase in cholesterol and sphingomyelin levels of the aged cerebella. Finally, we used the Martini coarse-grained model to conduct molecular dynamics simulations, from which we observed that membrane cholesterol concentrations can dictate whether the GABABR tail domains physically establish G protein-independent contacts with a transporter, and the timing when those associations eventually occur. Taken together, our findings illustrate how age-related alterations in membrane cholesterol levels affect protein-protein interactions, and how they could play a crucial role in regulating GABABR\'s interactome-mediated signaling.
UNASSIGNED: This study elucidates age-related changes in cerebellar GABAB receptors (GABABRs),
KCC2, and plasma membrane lipids, shedding light on mechanisms underlying neurological decline. Molecular dynamics simulations reveal how membrane lipids influence protein-protein interactions, offering insights into age-related neurodegeneration. The findings underscore the broader impact of cerebellar aging on motor functions, cognition, and emotional processing in the elderly. By elucidating plasma membrane regulation and GABAergic dynamics, this research lays the groundwork for understanding aging-related neurological disorders and inspires further investigation into therapeutic interventions.