The influence of membrane cholesterol on GABAA currents in a acutely dissociated rat hippocampal neurones

Cholesterol has been shown to act as a modulator of many membrane proteins and this thesis extends these observations to the GABAA receptor. Since the neuroactive steroids are structurally related to cholesterol and are known to modulate the function of the GABAA receptor, it was hypothesised that membrane cholesterol might interact with the recognition site for neuroactive steroids on the GABAA receptor. Acutely dissociated rat hippocampal neurones were enriched with cholesterol by incubation with either liposomes containing cholesterol or methyl-β-cyclodextrin complexed with cholesterol. Omission of the cholesterol from these carrier systems resulted in depletion of the neuronal cholesterol. Enrichments to 235% of control and depletions to 54% of control could be achieved whilst maintaining neuronal viability. Electrophysiological responses of the GABAA receptor to GABA were observed with the whole-cell application of the patch clamp technique. Both depletion and enrichment of membrane cholesterol reduced the sensitivity of the GABAA receptor to GABA but only enrichment reduced the potency of the competitive antagonist bicuculline methochloride. The antagonistic effect of picrotoxinin was not affected by either enrichment or depletion of cholesterol. The effects of the GABAA potentiators propofol, flunitrazepam and pentobarbital sodium were enhanced by cholesterol enrichment and could also be enhanced by enrichment with epicholesterol. Cholesterol depletion did not affect these GABA modulators. The cholesterol dependence of the positive (pregnanolone, allopregnanolone and alphaxalone) and negative (pregnenolone sulphate) modulatory neuroactive steroids was quite distinct, the effect of all these steroids being reduced by cholesterol enrichment and enhanced by depletion. The results suggest that membrane cholesterol may act as an antagonist of the neuroactive steroids at their recognition sites. Epicholesterol seems to act as a partial agonist at this cholesterol site. Altogether, the evidence favours sites of action of the neuroactive steroids on the transmembrane segments of the GABAA receptor at the level of the outer leaflet of the membrane bilayer