Synaptosomal membranes-based Langmuir-Blodgett films. A platform for studies on gamma.aminobutyric acid type A receptor binding properties

TURINA AV; CLOP, PD; PERILLO MA

LANGMUIR

AMER CHEMICAL SOC

Lugar: Washington; Año: 2015 vol. 31 p. 1782 - 1801

0743-7463

In this work we used Langmuir Blodgett films (LB) as model membranes to study the effect of molecular packing on the flunitrazepam (FNZ) accessibility to the binding sites at the GABAA receptor (GABAA-R). Ligand binding data were correlated with film topography analysis by atomic force microscopy images (AFM) and SDS-PAGE. Langmuir films (LF) were prepared by the spreading of synaptosomal membranes (SM) from bovine brain cortex at the air?water interface. LBs were obtained by the transference, at 15 or 35 mN/m constant surface pressure (π), of one (LB15/1c and LB35/1c) or two (LB35/2c) LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference was performed in a serial manner, which allowed the accumulation of a great number of samples. SDS-PAGE clearly showed a 55 kDa band characteristic of GABAA-R subunits. Detrended fluctuation analysis of topographic data from AFM images exhibited a single slope value (self-similarity parameter α) in CONglass and a discontinuous slope change in the α value at an autocorrelation length of ∼100 nm in all LB samples, supporting the LF transference to the substrate. AFM images of CONglass and LB15/1c exhibited roughness and average heights that were similar between measurements and significantly lower than those of LB35/1c and LB35/2c, suggesting that the substrate coverage in the latter was more stable than in LB15/1c. While [3H]FNZ binding in LB15/1c did not reach saturation, in LB35/1c the binding kinetics became sigmoid with a binding affinity lower than in the SM suspension. Our results highlight the π dependence of both binding and topological data and call to mind the receptor mechanosensitivity. Thus, LB films provide a tool for bionanosensing GABAA-R ligand binding as well as GABAA-R activity modulation induced by the environmental supramolecular organization.