Bennett, Peter John; (1997) The influence of membrane cholesterol on the rat GABAA receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The GABAA receptor is an integral membrane protein surrounded by synaptic membrane lipids that may exert a biological control upon the receptor. An important component of the lipid milieu is cholesterol that constitutes over 20% of the membrane lipids which may interact with the neurosteroid binding sites of the GABAAR. TO investigate this possibility synaptic membranes were enriched with cholesterol by incubating with liposomes comprising 50 phosphatidylcholine : 50 cholesterol in the presence of 1% BSA. The effects of cholesterol and modulatory drugs on the GABAA channel were assessed via changes induced in [3H]-FNZ, [3H]-muscimol or [3H]-TBOB binding. Cholesterol enrichment did not affect the affinity of FNZ, muscimol or TBOB for their binding sites; however, the enhancement of [3H]-FNZ binding by pregnanolone, muscimol or propofol; the modulation of [3H]-muscimol by propofol and the modulation of [3H]-TBOB binding by FNZ, pregnanolone, muscimol or propofol were affected. In enriched cerebral cortex membranes, the potency of pregnanolone to enhance [3H]-FNZ binding was reduced. By contrast, in membranes from spinal cord, the potency of pregnanolone was increased following cholesterol enrichment. In membranes from cerebellum, there was little overall change in pregnanolone potency although the effects of threshold concentrations were increased. The enhancement of [3H]-FNZ binding by propofol was reduced and the potency of muscimol to potentiate [3H]-FNZ binding was increased upon enrichment of whole brain, cerebral cortex and spinal cord membranes. Enhancement of [3H]-muscimol binding by propofol in whole brain membranes was reduced upon cholesterol enrichment. In whole brain membranes modulation of [3H]-TBOB binding by FNZ, pregnanolone, muscimol or propofol was reduced upon cholesterol enrichment. 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 modulatory sites on the GABAA channel. The detailed pattern of influence on the enhancement of [3H]-FNZ binding by pregnanolone depended on the region of CNS and may be related to the subunit composition of the GABAA channels present.

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