The influence of membrane cholesterol on the GABAA receptor.

1. Neurosteroids such as pregnanolone have been established as potent modulators of the GABAA receptor in both electrophysiological and binding studies. Since cholesterol is present in substantial amounts in the neuronal membranes, we have sought evidence for possible interactions of cholesterol with the neurosteroid site and more generally, with the GABAA channel. 2. Synaptosomal membranes were prepared from rat whole brain, cerebral cortex, cerebellum and spinal cord. These membranes were enriched with cholesterol to about double the original level by incubation with liposomes comprised of 50 phosphatidylcholine: 50 cholesterol in the presence of 1% BSA. The additional cholesterol formed a homogeneous mixture with the endogenous cholesterol. 3. The effects of cholesterol and modulatory drugs on the GABAA channel were assessed from the changes induced in [3H]-flunitrazepam (FNZ) binding. Cholesterol enrichment did not affect FNZ binding itself; however, the enhancement of [3H]-FNZ binding by pregnanolone was affected. In membranes from cerebral cortex, the potency of pregnanolone was reduced by a factor of 3.2 following cholesterol enrichment. By contrast, in membranes from spinal cord, the potency of pregnanolone was increased by a factor of 8.4 following cholesterol enrichment. In membranes from cerebellum, there was little overall change in pregnanolone potency although the effects of threshold concentrations were increased. 4. The enhancement of [3H]-FNZ binding by propofol in whole brain membranes was reduced in cholesterol-enriched membranes, similar to the effects of pregnanolone. Experiments with muscimol resulted in an increase in its potency as a potentiator of [3H]-FNZ binding, following cholesterol enrichment. 5. These results provide little evidence for a selective competition between cholesterol and pregnanolone at its binding site. Rather, they suggest an influence of membrane cholesterol on the functional coupling between the benzodiazepine site and the other specific drug sites on the GABAA channel. The detailed pattern of influence depended upon the region of CNS and may be related to the subunit composition of the GABAA channels present.