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

ANAHÍ V. TURINA; PEDRO D. CLOP; MARIA A. PERILLO

LANGMUIR

AMER CHEMICAL SOC

Lugar: Washington; Año: 2015 vol. 31 p. 1792 - 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 ofsynaptosomal membranes (SM) from bovine brain cortex at the air waterinterface. LBs were obtained by the transference, at 15 or 35 mN/m constantsurface pressure (pi), of one (LB15/1c and LB35/1c) or two (LB35/2c)LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference wasperformed in a serial manner, which allowed the accumulation of a great numberof 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 alpha)in CONglass and a discontinuous slope change in the alpha value at an autocorrelation length of near to100 nm in all LB samples, supporting the LF transference to thesubstrate. 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]FNZbinding 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. Ourresults highlight the pi dependence of both binding and topological dataand call to mind the receptor mechanosensitivity. Thus, LB films provide a toolfor bionanosensing GABAA-R ligand binding as well as GABAA-R activitymodulation induced by the environmental supramolecular organization.