Coupling Between GABA^sub A^-R Ligand-Binding Activity and Membrane Organization in [beta]-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

• Published in: Cell biochemistry and biophysics Vol. 63; no. 1; p. 17
• Main Authors: Turina, Anahí V, Schreier, Shirley, Perillo, María A
• Format: Journal Article
• Language: English
• Published: Totowa Springer Nature B.V 01.05.2012
• Subjects: Anisotropy; Centrifugation; Cooling; Fluorescence; Heating; Membranes
• ISSN: 1085-9195; 1559-0283
• DOI: 10.1007/s12013-012-9338-1

Correlations between GABA^sub A^ receptor (GABA^sub A^-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with β-cyclodextrin (β-CD). The mere pre-incubation (PI) at 37°C accompanying the β-CD treatment was an underlying source of perturbations increasing [^sup 3^H]-FNZ maximal binding (70%) and K ^sub d^ (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A ^sub DPH^) in SMs submitted to a heating-cooling cycle (4-37-4°C) with A ^sub DPH,heating^ < A ^sub DPH,cooling^. Compared with PI samples, the β-CD treatment reduced B ^sub max^ by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A ^sub TMA-DPH^. PI, but not β-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with β-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, β-CD is not completely eliminated from the system through centrifugation washings. It was concluded that β-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual β-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA^sub A^-R density, relative to untreated samples.[PUBLICATION ABSTRACT]