Dynamics of GABABR signaling: influence of cholesterol and aging

Abstract

GABA B receptors (GABABRs) are obligatory heterodimers which belong to the superfamily of G protein-coupled receptors (GPCRs). Age-related changes in membrane cholesterol levels modulate membrane fluidity, which in-turn influences GPCRs´ localization and function. We studied the GABABR and also a transmembrane transporter structurally homologous to KCC2. To characterize transient conformational changes over time, molecular dynamics simulations were performed using a neuronal plasma membrane (PM) model. Two different membrane cholesterol levels were evaluated: 45% and 10%, which intend to resemble the composition of adult and aged neuronal PMs, respectively. For experimental verification in both young and aged cerebella, we determined protein expression and distribution, and we assessed whether the two proteins interact with each other in vivo. Techniques were: western blots (WB), co-immunoprecipitation assays, and multiple immunolabeling followed by confocal microscopy. Our results suggest that the expression and spatial distribution of both proteins change as the cerebellum grows older. Based on our in silico analyses, we infer that a G protein-independent interaction does occur. Also, we confirmed that the two proteins are part of the same complex in the cerebellum. As our simulations indicate, we propose that the underlying mechanism implies transient conformational changes, which are highly dependent on cholesterol levels and are therefore affected by the aging process.