EMAIL THIS PAGE TO A FRIEND

Cell biochemistry and biophysics

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.


PMID 22311134

Abstract

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.