Coupling between GABA(A)-R ligand-binding activity and membrane organization in β-cyclodextrin-treated synaptosomal membranes from bovine brain cortex: new insights from EPR experiments.
Correlations between GABA(A) receptor (GABA(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 [(3)H]-FNZ maximal binding (70%) and K                         (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                         (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4°C) with A                         (DPH,heating) < A                         (DPH,cooling). Compared with PI samples, the β-CD treatment reduced B                         (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                         (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(A)-R density, relative to untreated samples.
IIBYT, CONICET - Biofísica-Química, Departamento de Química, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina. aturina@efn.uncor.edu
Anahí V
AV
Turina
Shirley
S
Schreier
María A
MA
Perillo
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005445:Flunitrazepam / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding / Q000187:drug effects; D011963:Receptors, GABA-A / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D002273:Carcinogens; D008024:Ligands; D011963:Receptors, GABA-A; D047392:beta-Cyclodextrins; D005445:Flunitrazepam; D002784:Cholesterol; C031215:betadex