Obtention and characterization of E8C12-resistant and soluble membranes enriched with GABAA-R

COLMANO, NICOLÁS; SÁNCHEZ M; TURINA, ANAHÍ V.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Membrane protein purification and characterization requires membrane solubilization by detergents. Membrane components tend to aggregate so their dispersion is achieved by detergents replacing other hydrophobic components, such as lipids interacting with hydrophobic surfaces of membrane proteins. The abundance of transmembrane proteins is considerably lower than that of soluble proteins so the use of detergents to increase their availability becomes a must. Dodecanoyl-polyoxyethylene (E8C12) is a UV-invisible, nonionic detergent with properties similar to Triton X-100 with a proven ability to solubilize and purify membrane proteins. Since GABAA-R was found enriched in postsynaptic insoluble material of synaptosomes treated with 1% Triton X-100, in this work we evaluate the capacity of E8C12 detergent to generate a GABAA-R enriched fraction. Soluble and resistant fractions (DSM & DRM, respectively) were obtained from 0.32%(w/v) E8C12 treated membranes. They presented differences in the percentages of phospholipids, cholesterol, and proteins compared with the NM, being DRMs the most cholesterol-enriched ones. Compression isotherms at the air-water interface and their compressional modulus indicated that the membranes exhibit a liquid behavior throughout the whole isotherms. DRM films were more stable, expanded, and compressible than NM. Compressional modulus showed the typical two-dimensional transitions in NM at πt ⁓15mN/m whereas in DRM, a valley discontinuity was observed. DRM films transitioned two-dimensionally at π=35mN/m and depicted similar Cs-1 values to those of the collapse peak (⁓49 mN/m). The presence and abundance of the GABAA-R in the films were evidenced by western blotting and visualized through epifluorescence microscopy of hydrophobic silica transferred via Langmuir-Schaefer (LS) method of double-labeled membranes with DiI-C18 and Anti-subunit α1-GABAA-R revealed with an Alexa Fluor 350 secondary antibody. The effects of lateral pressure on the evolution of DiI-C18 grey areas in NM were similar to what was reported previously. The analysis showed, at 15 mN/m, little differences between NM and DRMs, however, at 35mN/m the DiI-enriched areas (LGA & BGA) were considerably higher for DRMs. The images of immunolabeled membranes evinced the presence of the receptor in all analyzed fractions and suggests that the DRM fraction is slightly enriched in GABAA-R. Western blot results allowed us to make a semiquantitative measurement of its presence.