Membrane treatment with beta-CD: effects on synaptosomal membranes organization and R-GABAA activity.

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

Montevideo, Uruguay

Congreso; 5th Southern Cone Biophysical Congress and 34th Annual Meeting of the Argentinean Biophysical Society; 2007

In the present work we investigated the effectiveness b-cyclodextrines (b-CD) to modify the cholesterol (Chol) content of synaptosomal membranes (SM) and the consequence of this treatment on the activity of GABAA receptor (R-GABAA). The latter was evaluated through the kinetics of [3H]-FNZ specific binding. The b-CD treatment induce a decrement in the maximal binding value Bmax (from 2120 to 2009 fmol/mg prot.), compared with a SM that suffers the pre-incubation necessary to the b-CD extract Chol but in absence of b-CD, without effect on the binding affinity. By the means of chemical methods, we demonstrated a 45% decrement in the relative Chol content of SM treated that was correlated with a diminution of the supramolecular organization as shown by EPR experiments. We measured a decrement in the order parameter S (from 0.762 to 0.715) of 5-SASL, that sense the environment in the C5 region. In addition, the EPR experiments allowed to demonstrate that, contrary to which is normally assumed, the b-CD is not totally eliminated of the system by centrifugation washings as well as the capacity of b-CD to interact with fatty acids, represented by the spin labels probes. The effect of the pre-incubation at 37ºC that accompanied the treatment with b-CD was a secondary source of perturbations in SM organization exhibiting a decrease in the h+1/ho values of 12-SASL.  According to previous reports (García et al, 2002), the pre-incubation induced an increment, from 659.4 to 2120 fmol/mg prot., in the Bmax value, without effecting the binding affinity. On the other hand, we observed a hysteresis in the DPH fluorescence anisotropy in SM submitted to a heating/cooling cycle (4ºC-37ºC-4ºC), a procedure that accompanies the b-CD treatment. This result suggested that the pre-incubation improve the hydratation of the liofilized SM. Nevertheless, other changes must be happening since that behavior also was observed in non-liofilized membranes (García et al., 2002).    The absence of possible artifacts induced by the b-CD on Chol and protein quantification as well as on the retention of [3H]-FNZ complexed with b-CD was demonstrated.