Coupling between GABAA-R activity and membrane organziation in cholesterol-depleted synaptosomal membranes from bovine brain cortex. New insights from EPR experiments.

TURINA A.V.; SCHREIER S.; PERILLO M.A.

CELL BIOCHEMISTRY AND BIOPHYSICS

HUMANA PRESS INC

Año: 2012 vol. 63 p. 17 - 33

1085-9195

Correlations between GABAA-receptor (GABAA-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with b-cyclodextrin (b-CD). The mere pre-incubation (PI) at 37ºC accompanying the b-CD treatment was an underlying source of perturbations increasing [3H]-FNZ maximal binding (70%) and Kd (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 (ADPH) in SMs submitted to a heating-cooling cycle (4ºC-37ºC-4ºC) with ADPH,heating<ADPH,cooling. Compared with PI-samples, the b-CD treatment reduced Bmax 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 ATMA-DPH. PI, but not b-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with b-CD-, SM-partitioned and free in solution showed that, contrary to what is usually assumed, b-CD is not completely eliminated from the system through centrifugation washings. It was concluded that, b-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 b-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 GABAA-R density, relative to untreated samples