Haneef, Fouzia; (2001) Influence of membrane cholesterol on neurosteroid modulation of BNZ/GABAA receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cholesterol has been shown to influence the functional properties of many receptors, including the GABAA receptor. The influence of membrane cholesterol on the modulatory site of the GABAA receptor was investigated via changes induced on [³H]-flunitrazepam (FNZ) binding. The effect of cholesterol on [³H]-FNZ binding modulated by neurosteroids and the non-steroid propofol was also investigated in native receptors of rat cerebral cortex as well as in recombinant GABAA receptors expressed in fibroblasts. Cholesterol enrichment (181-191% of control) and cholesterol depletion (43-49% of control) in cortical membrane fragments was achieved with incubation with cholesterol-methyl-β-cyclodextrin inclusion complex and methyl-β-cyclodextrin, respectively. A high and a low affinity component for [³H]-FNZ binding were identified in control and cholesterol enriched cortical membranes, and in control fibroblast membranes. The two components were believed to be coupled to the GABAA receptor. Both cholesterol enrichment and cholesterol depletion affected the high affinity [³H]-FNZ binding in cortical membranes. The modulatory action of neurosteroids (pregnanolone, allopregnanolone and alphaxalone) and propofol on [³H]-FNZ binding was increased to a similar extent in control, cholesterol enriched and cholesterol depleted cortical membranes. Pregnanolone also increased the [³H]-FNZ binding in control fibroblast membranes. It was proposed that neurosteroids and propofol enhance [³H]-FNZ binding by interfering with the low affinity component. Reducing the temperature from 21°C to 4°C affected the [³H]-FNZ binding affinity in control and cholesterol depleted cortical membranes. Similar results were also obtained in control fibroblast membranes. Epicholesterol mimicked the effects of cholesterol on high affinity [³H]-FNZ binding. The effects of coprostanol were similar to cholesterol depletion and the effects of epicoprostanol showed pregnanolone-like action on the GABAA receptor. All three sterols, unlike cholesterol, blocked the action of pregnanolone in potentiating [³H]-FNZ binding. Overall, it appears that the cholesterol and the related sterols studied have a variety of effects on pregnanolone with regard to its ability to modulate [³H]-FNZ binding.

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