The School of Pharmacy University of London
THE INFLUENCE OF MEMBRANE CHOLESTEROL
ON GABAa c u r r e n t s IN ACUTELY DISSOCIATED RAT HIPPOCAMPAL NEURONES
Thongchai Sooksawate B.Sc. in Pharm., M.Sc.
Department of Pharmacology, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WCIN lAX UK
A thesis submitted for the Degree of Doctor of Philosophy, University of London
1999 ProQuest Number: 10104210
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract
Cholesterol has been shown to act as a modulator of many membrane proteins and this thesis extends these observations to the GABA a receptor. Since the neuroactive steroids are structurally related to cholesterol and are known to modulate the function of the GAB A A receptor, it was hypothesised that membrane cholesterol might interact with the recognition site for neuroactive steroids on the GABA a receptor.
Acutely dissociated rat hippocampal neurones were enriched with cholesterol by incubation with either liposomes containing cholesterol or methyl-P-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 GABA a receptor to G ABA were observed with the whole-cell application of the patch clamp technique. Both depletion and enrichment of membrane cholesterol reduced the sensitivity of the GABA a receptor to G ABA 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 GABA a 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 GAB A 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 GABA a receptor at the level of the outer leaflet of the membrane bilayer.
The Influence of Membrane Cholesterol on GABA^ Currents page 2 Contents
Page
Abstract 2 Contents 3 List of Figures 8 List of Tables 14 List of Publications 15
Chapter 1: General Introduction 16
1.1 Plasma membrane of animal cells 17 1.2 Cholesterol and biological membranes 18 1.2.1 Cholesterol location in the cells 18 1.2.2 Cholesterol effects on the physical properties of the 19 membranes 1.2.3 Cholesterol effects on the functions of membrane proteins 19 1.2.4 Cholesterol modulation of ligand-gated ion channels 20
1.3 GAB A and the GABA a receptor 22
1.3.1 Molecular structure of the GABA a receptor 22
1.3.2 Pharmacology of the GABA a receptor 26
1.4 Steroid modulation of the GABA a receptor 27
1.4.1 Neuroactive steroid potentiation of GABA a receptor 27 activation 1.4.2 Structure-activity relationship for neuroactive steroid 29
potentiation of GABA a receptor activation 1.4.3 Sites of action for GAB A potentiation by neuroactive 32 steroids
1.4.4 Neuroactive steroid antagonism of the GABA a receptor 32
The Influence o f Membrane Cholesterol onGABA a,Currents page 3 Page
1.5 Effect of membrane lipids on the GABA a receptor 33 1.6 Aims and objectives 33
Chapter 2: Materials and Methods 35
2.1 Materials and Methods 36 2.1.1 Liposome preparation 36 2.1.2 Preparation of methyl-P-cyclodextrin solution or 36 its cholesterol inclusion complex 2.1.3 Preparation of epicholesterol-methyl-P-cyclodextrin 37 inclusion complex 2.1.4 Dissociation of hippocampal neurones 37 2.1.5 Manipulation of membrane cholesterol using liposomes 38 2.1.6 Manipulation of membrane cholesterol using methyl-P- 39 cyclodextrin or its cholesterol inclusion complex 2.1.7 Incubation of the neurones with epicholesterol using 40 epicholesterol-methyl-p-cyclodextrin inclusion complex 2.1.8 Cholesterol and protein assays of hippocampal neurones 41 2.1.9 Electrophysiological measurements 43 2.1.10 Drugs and chemicals 43 2.1.11 Data analysis 45 2.2 Discussion 46
The Influence of Membrane Cholesterol on GABA^ Currents page 4 Page
Chapter 3: Manipulation of Membrane Cholesterol 50
3.1 Introduction 51 3.2 Experimental protocols and results 52 3.2.1 The viability of acutely dissociated hippocampal neurones 52 3.2.2 Alterations of membrane cholesterol content by liposomes 52
3.2.3 Alterations of membrane cholesterol content by methyl-(3- 56 cyclodextrin and its cholesterol inclusion complex 3.2.4 Restoration of membrane cholesterol following depletion 59 3.3 Discussion 61
Chapter 4: Effect of Membrane Cholesterol on the Sensitivity of 64
GABAa Receptors to GABA
4.1 Introduction 65
4.2 Experimental protocol and results 6 6
4.2.1 Effect of membrane cholesterol on the GABA ECso 6 6 4.2.2 Restoration of membrane cholesterol and GABA EC50 74 values following depletion 4.2.3 Effect of membrane cholesterol on the current-voltage 74 relationship
4.2.4 Effect of acute application of cholesterol on GABA a 77 currents 4.3 Discussion 79
The Influence of Membrane Cholesterol on GABA^ Currents page 5 Page
Chapter 5: Effect of Membrane Cholesterol on the Potentiation of 81
GABAa Currents by Neuroactive Steroids
5.1 Introduction 82 5.2 Experimental protocols and results 84 5.2.1 Effect of membrane cholesterol on the potentiation of 84
GABA a currents by neuroactive steroids
5.2.2 Restoration of membrane cholesterol and GABA EC 50 97 ratios following depletion 5.2.3 Effect of acute application of cholesterol on the 97
potentiation of GABA a currents by pregnanolone 5.3 Discussion 100
Chapter 6: Effect of Membrane Cholesterol on the Modulation of 102
GABAa Currents by Other Drugs
6.1 Introduction 103 6.2 Experimental protocols and results 104 6.2.1 Effect of membrane cholesterol on the potentiation of 104 GABAa currents by drugs 6.2.2 Effect of membrane cholesterol on the antagonism of 112 GABAa currents by drugs 6.3 Discussion 120
The Influence o f Membrane Cholesterol on GABA^ Currents page 6 Page
Chapter 7: Effect of Incubation with Epicholesterol on the 122
potentiation of GABAa Currents by Drugs
7.1 Introduction 123 7.2 Experimental protocols and results 125 7.2.1 Effect of incubation with epicholesterol on the 125
sensitivity of the GABA a receptor to GABA 7.2.2 Effect of incubation with epicholesterol on the 128
potentiation of GABA a currents by pregnanolone 7.2.3 Effect of incubation with epicholesterol on the 131
potentiation of GABA a currents by other drugs 7.3 Discussion 133
Chapter 8 ; General Discussion 135
8.1 Cholesterol as a modulator of the GABA a receptor 136 8.1.1 The influence of membrane cholesterol on the effect of 136 GABA
8 .1 .2 Cholesterol interaction with modulators of the GABAa 138 receptor 8.2 Comparison with [^H]flunitrazepam binding studies 139 8.3 Sites of cholesterol-neuroactive steroid interaction 140
8.4 Implications of cholesterol interaction with the GABA a receptor 143 8.5 Future perspectives 145
Acknowledgements 146 References 147
The Influence o f Membrane Cholesterol on GABA^ Currents page 7 List of Figures
Figure Page
1 .1 Model of the GABAa receptor. 23 1.2 Outline of the synthetic pathway for neuroactive steroid synthesis in 28 the central nervous system. 1.3 The lettering of the steroid rings and numbering of the carbon atoms in 31
the steroid 5p-pregnan-3a-ol-20-one (pregnanolone).
2.1 Standard curve of cholesterol measured by cholesterol diagnostic kit. 42
3.1 Photomicrographs of acutely dissociated hippocampal neurones 53 from 10-16 days old Wistar rat brains before and after membrane cholesterol alterations. 3.2 Alterations of membrane cholesterol by liposomes in acutely 55 dissociated rat hippocampal neurones. 3.3 Alterations of membrane cholesterol by methyl-P-cyclodextrin 57 in acutely dissociated rat hippocampal neurones. 3.4 Restoration of membrane cholesterol following depletion. 60
4.1 The effect of membrane cholesterol alterations by liposomes 67
on GABA a currents in acutely dissociated rat hippocampal neurones.
4.2 The effect of membrane cholesterol alterations by liposomes 6 8 on GABA concentration-response relationship. 4.3 The effect of membrane cholesterol alterations by liposomes 69
on the EC 50 values for GABA concentration-response relationship
The Influence o f Membrane Cholesterol on GABA^ Currents page 8 Figure Page
4.4 The effect of membrane cholesterol alterations by 71
methyl-P-cyclodextrin on GABA a currents in acutely dissociated rat hippocampal neurones.
4.5 The effect of membrane cholesterol alterations by methyl-p- 72 cyclodextrin on GABA concentration-response relationship.
4.6 The effect of membrane cholesterol alterations by methyl-P- 73
cyclodextrin on the EC 50 values for GABA concentration- response relationship.
4.7 Restoration of membrane cholesterol and GABA EC 50 values 75 following cholesterol depletion. 4.8 The effect of membrane cholesterol alterations by methyl-P- 76
cyclodextrin on the GABA a receptor current-voltage relationship. 4.9 The effect of acute application of cholesterol on the GABA 78 concentration-response curves.
5.1 The effect of membrane cholesterol alterations by liposomes on the 85
potentiation of GABA a currents by 1 |xM pregnanolone. 5.2 The effect of membrane cholesterol alterations by liposomes on 86 the concentration-response relationship for the potentiation of
GABA a currents by 1 pM pregnanolone. 5.3 The effect of membrane cholesterol alterations by liposomes on 87 the concentration-response relationship for the potentiation of
GABA a currents by 1 pM alphaxalone.
The Influence of Membrane Cholesterol onGABA a Currents page 9 Figure Page
5.4 The effect of membrane cholesterol alterations by liposomes on 8 8
the concentration-response relationship for the potentiation of GABA a currents by 0.3 }xM pregnanolone and 1 pM allopregnanolone.
5.5 The effect of membrane cholesterol alterations by methyl-p- 92
cyclodextrin on the potentiation of GABAa currents by 1 pM pregnanolone.
5.6 The effect of membrane cholesterol alterations by methyl-p- 93 cyclodextrin on the concentration-response relationship for the
potentiation of GABAa currents by 1 pM pregnanolone. 5.7 The effect of membrane cholesterol alterations by methyl-P- 94 cyclodextrin on the concentration-response relationship for the
potentiation of GABAa currents by 1 pM alphaxalone.
5.8 The effect of membrane cholesterol alterations by methyl-p- 95
cyclodextrin on the GABA EC 50 ratios for the potentiation of GABAa currents by IpM pregnanolone. 5.9 Restoration of membrane cholesterol and GAB A EC50 ratios 98
for the potentiation of GABA a currents by IpM pregnanolone in cholesterol depleted neurones (56% of control). 5.10 The effect acute application of cholesterol on the concentration- 99
response curves for the potentiation of GABA a currents by 1 pM pregnanolone.
6.1 The effect of membrane cholesterol alterations by methyl-p- 105
cyclodextrin on the potentiation of GABA a currents by 5 pM propofol.
The Influence of Membrane Cholesterol on GABA^ Currents page 10 Figure Page
6.2 The effect of membrane cholesterol alterations by methyl-P- 106 cyclodextrin on the concentration-response relationship for the
potentiation of GABA a currents by 5 ^iM propofol. 6.3 The effect of membrane cholesterol alterations by liposomes on 108 the concentration-response relationship for the potentiation of
GAB A A currents by 5 \iM propofol. 6.4 The effect of membrane cholesterol alterations by methyl-^- 110 cyclodextrin on the concentration-response relationship for the
potentiation of GABA a currents by 20 }xM pentobarbital sodium.
6.5 The effect of membrane cholesterol alterations by methyl-^- 111 cyclodextrin on the concentration-response relationship for the
potentiation of GABA a currents by 10 |liM flunitrazepam.
6.6 The effect of membrane cholesterol alterations by methyl-(3- 113
cyclodextrin on the antagonism of GABA a currents by 5 |xM bicuculline methochloride. 6.7 The effect of membrane cholesterol alterations by methyl-p- 114 cyclodextrin on the concentration-response relationship for the
antagonism of GABA a currents by 5 |.iM bicuculline methochloride. 6.8 The effect of membrane cholesterol alterations by methyl-P- 115 cyclodextrin on the concentration-response relationship for the
antagonism of GABA a currents by 10 )LiM picrotoxinin.
6.9 The effect of membrane cholesterol alterations by methyl-P- 117
cyclodextrin on the antagonism of GABA a currents by 20 pM pregnenolone sulphate.
The Influence of Membrane Cholesterol onGABA a Currents page 11 Figure Page
6.10 The effect of membrane cholesterol alterations by methyl-(3- 118 cyclodextrin on the concentration-response relationship for the
antagonism of GABAa currents by 2 0 p,M pregnenolone sulphate.
6.11 The effect of membrane cholesterol alterations by methyl-P- 119 cyclodextrin on the % reduction of the maximal GABA responses
caused by 2 0 piM pregnenolone sulphate.
7.1 The effect of epicholesterol incubation on the GABA 126 concentration-response relationship. 7.2 The effect of incubation of cholesterol-depleted (56% of control) 127
neurones with 0 .1 mM epicholesterol-methyl-P-cyclodextrin
complex for 5 and 20 min on the GABA EC 50 values. 7.3 The effect of incubation of the neurones with epicholesterol- 129 methyl-P-cyclodextrin complex for 20 min on the concentration-
response relationship for the potentiation of GABA a currents
by 1 |uiM pregnanolone. 7.4 The effect of incubation of cholesterol-depleted neurones (56% of 130 control) and control neurones with 0.10 and 0.15 mM epicholesterol-
methyl-P-cyclodextrin complex for 20 min on the GABA EC 50 ratios
for the potentiation of GABAa currents by 1 |liM pregnanolone. 7.5 The effect of incubation of the neurones with epicholesterol- 132 methyl-P-cyclodextrin complex for 20 min on the concentration-
response relationship for the potentiation of GABA a currents by 5 p.M propofol (A), 20 |iM pentobarbital sodium (B) and 10 ^iM flunitrazepam (C).
The Influence of Membrane Cholesterol on GABA^ Currents page 12 Figure Page
8.1 A model (not to scale) for the possible sites of interaction 141
between cholesterol and neuroactive steroids on the GABA a receptor protein.
The Influence o f Membrane Cholesterol on GABAa Currents page 13 List of Tables
Table Page
1.1 Distribution of the major GAB AA-receptor subtypes in the rat brain. 25
3.1 Alterations of membrane cholesterol by liposomes in acutely 54 dissociated rat hippocampal neurones. 3.2 Alterations of membrane cholesterol by methyl-P-cyclodextrin 58 in acutely dissociated rat hippocampal neurones.
5.1 The effect of membrane cholesterol alterations by asolectin 89
liposomes on GABA BC 50 ratios for the potentiation of GABAa currents by neuroactive steroids. 5.2 The effect of membrane cholesterol alterations by pure PC 90
liposomes on GABA BC50 ratios for the potentiation of GABA a currents by 1 |xM pregnanolone.
5.3 The effect of membrane cholesterol alterations by methyl-P- 96
cyclodextrin on GABA BC 50 ratios for the potentiation of GABAa currents by neuroactive steroids.
6.1 The effect of membrane cholesterol alterations by methyl-P- 107
cyclodextrin on GABA BC50 ratios for the modulation of GABA a currents by drugs.
7.1 The effect of incubation with 0.15 mM epicholesterol-methyl-P- 128
cyclodextrin complex for 20 min on the GABA BC 50 ratios
for the potentiation of GABA a currents by drugs.
The Influence of Membrane Cholesterol on GABA^ Currents page 14 List of Publications
Full article
Sooksawate T. and Simmonds M.A. (1998) Increased membrane cholesterol reduces the potentiation of GABAa currents by neurosteroids in dissociated hippocampal neurones. Neuropharmacology,'51: 1103-1110.
Abstracts
Sooksawate T. and Simmonds M.A. (1998) Effect of membrane cholesterol enrichment on the potentiation of GABAa currents by neurosteroids. Journal of Pharmacy and Pharmacology, 50: 213.
Sooksawate T. and Simmonds M.A. (1999) Effect of membrane cholesterol on the sensitivity of GABAa receptor to GABA in dissociated rat hippocampal neurones. Journal of Physiology, 515: 129P.
Sooksawate T. and Simmonds M.A. (1999) Effect of membrane cholesterol on the potentiation and inhibition of GABAa currents by neurosteroids in acutely dissociated rat hippocampal neurones. Journal of Physiology, 518: 154P-155P.
Sooksawate T. and Simmonds M.A. (1999) Effect of membrane cholesterol on the potentiation of GABAa currents by propofol in acutely dissociated rat hippocampal neurones. Journal of Pharmacy and Pharmacology, 51: 159.
The Influence of Membrane Cholesterol on GABA^ Currents page 15 CHAPTER 1
GENERAL INTRODUCTION
The Influence of Membrane Cholesterol onGABA a, Currents page 16 Chapter 1: General Introduction
1.1 Plasma membrane of animal cells
The plasma membrane of the cell defines the cell boundary. This membrane provides the reactive interface between the external environment and the cytoplasm. It controls the passage of materials into and out of the cell through its transport systems. It is the membrane that provides the initial response of the cell to all extracellular stimuli. In animals, the plasma membrane is constructed of amphipathic lipid made up of phospholipids, sphingolipids, glycolipids and cholesterol. Rat synaptic plasma membranes consist of phospholipids, representing about 74% of the total membrane lipids, and cholesterol about 19% of the total membrane lipids (Cotman et al., 1969). Proteins are also found in all biological membranes. The ratio by weight of protein to lipid in the plasma membranes is close to 1.0. This ratio is 0.9 in rat synaptic plasma membrane (Cotman et al., 1969).
The conventional picture of the biological membrane is that of Singer and Nicholson (1972) who suggested viewing the biological membrane as a fluid mosaic lipid bilayer in which the proteins are embedded or to which they are attached. The crucial property of this molecular assembly is the bilayer fluidity that assures sufficient lateral mobility of the membrane components to support biological function. Towards the cell interior, the lipid bilayer is attached to a cytoskeleton of extended polymeric filaments that are important for maintaining the shape of the cell. On the exterior side the membrane is covered by a glycocalyx that is made up by the carbohydrate moieties of membrane proteins and lipids. The interactions between the molecules of the bilayer, such as lipid-lipid, lipid-protein and lipid membrane- cytoskeletal, lead to a lateral organisation of the membrane often characterised by a substantial degree of static and dynamic membrane heterogeneity (Mouritsen & Biltonen, 1993). The lipid properties of the membrane are influenced by the presence of proteins. Likewise, the functional properties of membrane proteins are affected by the lipid environment.
The Influence of Membrane Cholesterol on GABA^ Currents page 17 Chapter 1: General Introduction
1.2 Cholesterol and biological membranes
1.2.1 Cholesterol location in the cells
Cholesterol is an essential component of mammalian cells. It plays many roles in the functioning of cell membranes as well as in the synthesis of steroids, bile acids and lipoproteins. Mammalian cells obtain cholesterol by de novo synthesis in their endoplasmic reticulum or by internalisation of low density lipoproteins. Cholesterol is disproportionately distributed among the membranes of cells. The plasma membrane contains as much as 90% of the cholesterol present within that cell (Yeagle, 1993). It has been suggested that this high concentration of cholesterol in the plasma membrane results in part from its close association with sphingomyelin (Liscum & Underwood, 1995). Within the plasma membrane, cholesterol may not be uniformly distributed but is instead localised into cholesterol-rich and cholesterol-poor domains (Liscum & Underwood, 1995). The transbilayer distribution of cholesterol is mostly reported to be enriched in the inner leaflet of the plasma membranes of several cell types; for example, human erythrocyte membranes (Schroeder et a i, 1991), mouse L-cell fibroblast plasma membranes (Jefferson et a l, 1991) and mouse synaptic plasma membranes (Igbavboa et a l, 1996). Mechanisms that regulate the transbilayer distribution of cholesterol are poorly understood (Wood et al., 1999). Cholesterol can move (flip-flop) between outer and inner leaflets of the membranes with a migration time ranging from a few minutes (ti /2 between 1-6 min) in biological membranes
(Schroeder et a l, 1991) to several hours (ti /2 between 9-38 hours) in liposomal membranes (Rodrigueza et al., 1995).
The Influence of Membrane Cholesterol onGABA a Currents page 18 Chapter 1: General Introduction
1.2.2 Cholesterol effects on the physical properties of the membranes
Cholesterol can affect the bulk properties of lipid bilayers such as membrane ordering, lateral diffusion of membrane lipids or proteins and temperature dependence of phospholipid phase transitions from the gel to the liquid crystalline state (Yeagle, 1991). It has a profound effect on the fluidity of phospholipid bilayers where it acts to increase the fluidity of lipids in the gel phase but decrease the fluidity of those lipids in the more physiologically relevant liquid-crystalline state (Yeagle, 1985). The presence of cholesterol in the membranes reduces the conformational flexibility of the hydrocarbon chains of the phospholipids, causing them to adopt an average conformation in which most carbon-carbon single bonds are in the trans (more ordered) configuration (Yeagle, 1991). The increase in ordering of the lipid hydrocarbon chains leads to more effective packing of the lipids in the bilayer and consequent reduction in the bilayer packing defects. Membrane permeability of small and uncharged molecules is reduced by this mechanism (Demel et a l, 1972). The structural requirements for a sterol to produce this special ordering effect include a planar steroid ring system, a p-hydroxyl at position 3, and a hydrophobic cholesterol-like tail at position 17 (Demel et a l, 1972).
1.2.3 Cholesterol effects on the functions of membrane proteins
Several membrane receptor proteins have been reported to depend upon cholesterol for their functional activities, for example, the nicotinic acetylcholine receptor (Jones and McNamee, 1988; Femandez-Ballester et a l, 1994), the oxytocin receptor and the brain cholecystokinin receptor (Gimpl et a l, 1997), and rhodopsin (Mitchell et a l, 1990). Other plasma membrane proteins have also been reported to be modulated by cholesterol, such as, the Na'^-K'^ ATPase, (Yeagle et a l, 1988; Vemuri & Philipson, 1989), the Na'^-Ca^^ exchanger (Vemuri & Philipson, 1989), the adenylate cyclase
The Influence of Membrane Cholesterol on GABA^ Currents page 19 Chapter 1: General Introduction
(Whetton et ah, 1983), the GABA transporter and the L-glutamic acid transporter from rat brain (Shouffani & Kanner, 1990). Cholesterol likely modulates the function of these membrane proteins by more than one mechanism, e.g. alteration of the bulk biophysical properties such as membrane fluidity and/or by a specific cholesterol- protein interaction (Yeagle, 1993). Cholesterol alters membrane physical properties by increasing the ordering of the lipid hydrocarbon chains of phospholipids and, thereby, reducing the free volume available to membrane proteins for the conformational changes that may be required for membrane protein function. Cholesterol also may bind to specific sites on the membrane proteins or have an influence at the lipid-protein interface to modulate protein function. Those latter mechanisms are likely to be more protein-specific.
1.2.4 Cholesterol modulation of ligand-gated ion channels
The nicotinic acetylcholine receptor is an integral membrane protein and the best- understood member of a gene superfamily of ligand-gated ion channels of which the GABAa receptor is a member. Until the work reported in this thesis commenced, it was the only ligand-gated ion channel on which the influence of membrane cholesterol had been studied. The work of Fong & McNamee (1986) revealed that cholesterol also plays an important modulatory role supporting the agonist-induced channel opening in the Torpedo nicotinic acetylcholine receptor. In native membranes, roughly 80% of the nicotinic acetylcholine receptors exist in a resting conformation that transiently conducts cations across the membrane in response to the binding of acetylcholine agonist (Ryan et al., 1996). The remaining 20% exist in a high affinity, channel-inactive, desensitised state that is stabilised by prolonged exposure to agonist. Cholesterol has been found to help stabilise this receptor in a resting conformation (Ryan et al., 1996). It also stabilises the secondary structure of the receptor’s transmembrane regions (Femandez-Ballester et al., 1994). There are.
The Influence of Membrane Cholesterol onGABA a Currents page 20 Chapter 1: General Introduction
broadly, three types of location at which cholesterol might-modulate this receptor (Jones & McNamee, 1988; Addona et al., 1998): in the lipid bilayer, the lipid-protein interface (annular sites), or on the protein itself (nonannular or interstitial sites). It is unlikely that cholesterol modulates this receptor via membrane fluidity alterations since the receptor activity was maintained either in low or high fluidity environments (Sunshine & McNamee, 1994). However, the binding sites of cholesterol on this receptor protein remain controversial. The use of a brominated steroid analogue in fluorescence studies revealed the binding sites of cholesterol at the nonannular or interstitial sites on the transmembrane region of this receptor (Jones & McNamee, 1988). On the contrary, the use of [’^^I]azido-cholesterol in photoaffinity labelling studies (Corbin et al., 1998) and cholesterol-containing phospholipid in fluorescence studies with an ethidium bromide fluorescence probe (Addona et al., 1998) indicated the binding domain for cholesterol to be at the lipid-protein interface of this receptor (annular sites) or very close to it (periannular sites). Since the acute application of cholesterol failed to affect the activities of the nicotinic acetylcholine receptor (Valera et al., 1992), the extracellular domains of this receptor are unlikely to be the binding sites for cholesterol.
No functional studies on interactions between membrane cholesterol and the GABA a receptor or any other member of the gene superfamily of ligand-gated ion channel have previously been reported, to my knowledge.
The Influence of Membrane Cholesterol on GABA^ Currents page 21 Chapter 1: General Introduction
1.3 GABA and the GABAa receptor y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Estimations of the percentage of all central synapses that use GABA as their transmitter range from, depending on the brain region, 20 to 50% (Sieghart, 1995). The actions of GABA are mediated by binding of GABA to two different receptor classes: GABA a (ionotropic) and GABAg
(metabotropic) receptors (Barnard et al, 1998). The activation of the GABA a receptor as a member of the ligand-gated ion channel superfamily is followed by the fast gating or opening of an integral chloride ion channel which results, in general, in the hyperpolarization of the neurones (Sieghart, 1995). The activation of the GABA b receptor as a member of the G-protein-linked superfamily is followed by an increased membrane conductance to Ca^"^ or via second messenger systems (Bowery, 1993) leading to a slower and more prolonged inhibition.
1.3.1 Molecular structure of the GABAa receptor
The molecular structure of the GABAa receptor is believed to be a pentamer of subunits that assemble to form a functional GABAa receptor-ion channel (Figure 1 .1) (Nayeem et a l, 1994). So far, a total of 19 GABAa receptor subunits belonging to several subunit classes (ai- 6 , pi-4, Y1-3, ô, e, n and pi. 3) have been cloned and sequenced from mammals (Shingai et a l, 1996; Davies et a l, 1997; Heblom & Kirkness, 1997; Barnard et a l, 1998). They exhibit about 60-80% amino acid sequence homology within a subunit class, but about 30-40% homology among the various subunit classes (Sieghart, 1995). Alternative splicing of GABAa receptor gene products has been identified in %, 0 6 , P2, P3, P4 and 72 subunits (Barnard et ah,
1998). The best characterised of these are the two forms of the72 subunit, 72 s (72Short) and 72 l (yzLong) (Whiting et a l, 1995).
The Influence of Membrane Cholesterol onGABA a Currents page 22 Chapter I: General Introduction
Figure 1.1 Model of the GABA a receptor. This receptor believed to be a pentamer of subunits that assemble to form a functional GABA a receptor-ion channel. Each of subunit consistslfour putative transmembrane domains numbered 1-4 (cylinders), a large extracellular domain at the N-terminal half of the polypeptide and a large cytoplasmic domain between TM3 and TM4. The TM2 may contribute to the formation of a hydrophilic lining of the channel pore. Modified from Macdonald & Olsen (1994).
Subunit sequence and topological structure
Plan view
nxtracellular
M em brane
Intraeel In tar
The Influence of Membrane Cholesterol on GABA^ Currents page 23 Chapter 1: General Introduction
The GABA a receptor subunits share 20-30% amino acid sequence homology with other ligand-gated ion channels, the nicotinic acetylcholine receptor, the glycine receptor and the 5-HT] receptor (Whiting et at., 1995). Each subunit of the GABA a receptor, and also other ligand-gated ion channels, {consists of four putative transmembrane hydrophobic segments (TMl-4), a large extracellular domain at the N-terminal half of the polypeptide and a large cytoplasmic domain between TM3 and TM4 (Figure 1.1) (Macdonald & Olsen, 1994; Whiting et al., 1995). The large hydrophilic N-terminal part contains several glycosylation sites and a loop formed by two conserved cysteines and is thought to contain the GABA recognition site. The large cytoplasmic loop contains possible phosphorylation sites. The TM2 may contribute to the formation of a hydrophilic lining of the channel pore.
Since five subunits are necessary for the formation of GABAa receptors, and there exist 19 different subunits, a large number of heteropentameric isoforms are theoretically possible. However, from what is known so far of excluded compositions and the restricted co-occurrence of subunits in certain cases, the true number is likely to be smaller than 800 combinations but still much more than for other known receptor subtypes (Barnard et ah, 1998). Some of the native GABAa receptors may have the stoichiometry 2a(32Y or 2a2py (with the y subunits in some cases being replaceable by 8 or by e) (Barnard et ah, 1998). As shown in Table 1.1, the largest population (43%) of GABAa receptor subtypes in rat brain is the aiPzYz subtype (McKeman & Whiting, 1996). Two other major populations are thejaiPz/sYz
(18%) and aspnYz/ys (primarily asPnYz) (17%) subtypes. The physiological roles of individual subtypes remain largely to be determined although some clear pharmacological distinctions have been described (for reviews see Macdonald & Olsen, 1994; Sieghart, 1995).
The Influence o f Membrane Cholesterol onGABA a Currents page 24 Chapter 1: General Introduction
Table 1.1 Distribution of the major GABAA-receptor subtypes in the rat brain (McKeman & Whiting, 1996).
Relative Subtype abundance in Location and putative function rat brain (%)
(X1P2Y2 43 Present in most brain areas. Localised to intemeurones in hippocampus and cortex, and cerebellar Purkinje cells 18 Present on spinal cord motor neurones and hippocampal pyramidal cells
% P nY 2/3 17 Present on cholinergic and monoaminergic neurones where they regulate acetylcholine and monoamine turnover
% P nY l 8 Present on Bergmann glia, nuclei of the limbic systems, and in pancreas
ctsPsYz/s 4 Predominantly present on hippocampal pyramidal cells
OePnY: 2 Present on cerebellar granule cells OôPnÔ 2 Present on cerebellar granule cells Ct4Pnô 3 Present in thalamus and hippocampal dentate gyrus Other minor subtypes 3 Present throughout the brain Location and function are listed w lere these have been investigated, and are not comprehensive. Other minor subtypes include aiOtePcYi, otiasPnYz, otiOtsPnYz and (XsPnYzS subtypes and are represented together as a small population.
The Influence of Membrane Cholesterol onGABA a Currents page 25 Chapter 1: General Introduction
1.3.2 Pharmacology of the GABAa receptor
The binding of GAB A to the GABA a receptor results in an increase in membrane conductance for chloride ions. Quantitative analysis of electrophysiological experiments suggests that at least two GAB A molecules must bind to the receptor for receptor activation (Sakmann et al., 1983). Opening of the chloride ion channels usually results in a reduced excitability of the neurones (Study & Barker, 1981; Sieghart, 1995). Nevertheless, in some cases, especially in developing neurones (Chen et ah, 1996), G ABA demonstrated excitatory actions. Apart from the natural transmitter, GAB A, there are several compounds that have been utilised as agonists at GAB A A receptor, including muscimol, isoguvacine, piperidine-4-sulphonic acid and 4,5,6,7-tetrahydroisoxazolol [5,4-c]pyridin-3-ol (THIP). Bicuculline acts as a competitive antagonist at the GABA a binding site. Some compounds such as picrotoxinin, pentylenetetrazol (PTZ) and f-butylbicyclophosphorothionate (TBPS) antagonise G ABA in a non-competitive manner (Sieghart, 1995). Penicillin represents another type of GABA a receptor antagonist. It does not compete for the agonist recognition site but appears to preferentially block GABA-activated open ion channels (Twyman et a l, 1991; Macdonald & Olsen, 1994).
Additionally, activation of the GABA a receptor can be positively modulated by a wide range of pharmacologically and clinically important drugs acting at distinct sites (for a review see Sieghart, 1995). These include benzodiazepines, barbiturates, neuroactive steroids, ethanol, propofol, loreclezole and Zn^"^. The effects of such drugs provide a sensitive discrimination between receptor subtypes and may also serve as markers of the influence of membrane lipids on receptor activity.
The Influence of Membrane Cholesterol on GABA^ Currents page 26 Chapter 1: General Introduction
1.4 Steroid modulation of the GABAa receptor
Neuroactive steroids are natural or synthetic steroids that rapidly modulate the excitability of neurones by action on membrane receptors such as those for inhibitory and (or) excitatory neurotransmitters (Paul & Purdy, 1992). The term “neurosteroids” applies to the steroids, the accumulation of which occurs in the nervous system independently, at least in part, of supply by the steroidogenic endocrine glands and which can be synthesised de novo in the nervous system from sterol precursors (Baulieu & Robel, 1998). (The term “neurosteroids” is often used more loosely than this in the literature when “neuroactive steroids” would be the more appropriate term.) Outline of the synthetic pathways for neuroactive neurosteroid synthesis in the central nervous system is shown in figure 1 .2 . Neurosteroids can be synthesised within neurones and glial cells (Lambert et al., 1995; Mensah-Nyagan et at., 1999).
Neuroactive steroids have been reported to have a potential efficacy to be a novel class of drugs for the therapeutic management of epilepsy, anxiety, insomnia, migraine and drug dependence (for a review see Gasior et al., 1999).
1.4.1 Neuroactive steroid potentiation of GABAa receptor activation
One of the major targets for neuroactive steroids is the GABAa receptor. The discovery of the potentiation of GAB A responses by neuroactive steroids was reported by Harrison and Simmonds in 1984, based on experiments investigating the electrophysiological effects of the steroidal anaesthetic alphaxalone (5a-pregnan-3a- o l-ll, 2 0 -dione).
The Influence of Membrane Cholesterol onGABA a Currents page 27 Chapter 1: General Introduction
Figure 1.2 Outline of the synthetic pathways for neuroactive steroid synthesis in the central nervous system. Solid arrows indicate that the biochemical reaction has been formally demonstrated. Dashed arrows indicate that that the occurrence of the enzyme has not yet been found in the nervous system. The steroids shown in the boxes have modulatory activity at the GABAa receptor. Modified from Mensah- Nyagan et al. (1999).
5a-pregnan-3a,21-diol-20 one (Allotetrahycirodeoxycorticosterone)
Cholesterol Deoxycorticosterone
Pregnenolone Progesterone ^5a-Pregnane- 5 a-Pregnan-3 a-ol-20-one \ 3,20 dione (Allopregnanolone)
Pregnenolone 5 P-Pregnan-3a-ol-20-one sulphate (Pregnanolone)
Dehydroepiandrosterone (DHEA)
DHEA sulphate Androsterone
The Influence o f Membrane Cholesterol on GABA^ Currents page 28 Chapter 1: General Introduction
At low concentrations (30-300 nM), alphaxalone, 3a-hydroxylated, 5a- or 5|3- reduced metabolites of progesterone and deoxycorticosterone markedly potentiate GABA-stimulated chloride conductance (Majewska et al., 1986; Peters et al., 1988;
Lambert et al., 1995). At high concentrations (>1 p.M) these compounds result in a direct opening of the GABA a receptor-associated chloride channel that could be inhibited by the GABA a receptor antagonist bicuculline (Cottrell et al., 1987; Lambert et al., 1990; Sieghart, 1995). From single channel recording, neuroactive steroids acting at the GABA a receptor increased both the frequency (i.e., a benzodiazepine-like effect) and duration (i.e., barbiturate-like effect) of chloride channel opening (Twyman & Macdonald, 1992; Macdonald & Olsen, 1994). Both electrophysiological and binding studies have suggested that neuroactive steroids can potentiate the action of GAB A at the GABA a receptors at a site of action distinct from those of benzodiazepines and barbiturates (Peters et al., 1988; Lambert et al., 1990; Simmonds, 1991; Lambert et al., 1995).
1.4.2 Structure-activity relationship for neuroactive steroid potentiation of
GABAa receptor activation
Neuroactive steroid modulation of the GABAa receptors shows clear structure- activity requirements (Harrison et al., 1987; Simmonds, 1991; Lambert et al., 1995; Zorumski et al., 1996; Hawkinson et al., 1998). The structural features of neuroactive steroids that are required for activity are listed below (see also Figure 1.3).
An a-hydroxyl group at position 3 is required, a 3|3-hydroxyl or 3-keto group resulting in little or no activity.
The Influence of Membrane Cholesterol onGABA a Currents page 29 Chapter 1: General Introduction
A keto group at either C20 of the pregnane steroid side chain or C17 of the androstane ring system is required. The reduction of the 20-keto group results in partial agonist activity.
The side chain at C l7 in the pregnane steroids must be in the P configuration for activity. The configuration at the 5 position, which markedly affects the shape of the steroid, has little effect on the potency of those pregnane steroids that have only 3a-hydroxyl and 20 keto substituents but, when an 11-keto group is also present,
the SP isomer becomes less potent whereas the potency of the 5a isomer is little affected. - A saturated ring system is not an absolute requirement for activity, 4-pregnene- 3a-ol-20-one exhibits similar activity to that of 5a-pregnan-3a-ol-20-one. - It is possible to confer water solubility upon pregnane steroids, by the introduction of a 2p-morpholinyl moiety (ORG20599), without substantial lost of activity. Introduction of appropriately para-substitued phenylethynyl groups at the 3P-
position of 5p-pregnane steroids, in particular, 3p-(p-acetylphenylethynyl)-5a-
pregnan-3a-ol-20-one (Co 152791), increases receptor potency of the steroids compared with allopregnanolone. - The (-k)-enantiomers of pregnane steroids and benz[e]indenes are required. The (-)-enantiomers were significantly less effective.
The progesterone metabolites pregnanolone (5P-pregnan-3a-ol-20-one) and allopregnanolone (5a-pregnan-3a-ol-20-one), the deoxycorticosterone metabolite 5a- pregnan-3a, 21-diol-20-one and the synthetic analogue alphaxalone (5a-pregnan-3a- o l-ll, 2 0 -dione) are among the most effective potentiators of GAB A at the GABAa receptor (Majewska et al, 1986; Harrison et ah, 1987; Peters et al., 1988; Lambert et a l, 1995).
The Influence of Membrane Cholesterol onGABA a Currents page 30 Chapter 1: General Introduction
Figure 1.3 The lettering of the steroid rings and numbering of the carbon atoms in the steroid 5p-pregnan-3a-ol-20-one (pregnanolone). By convention, the a configuration is that lying below the general plane of the ring system and the P configurations that projecting above the plane of the ring system. The orientation of the hydrogen at C5 of the reduced pregnane (21 carbon) and androstane (19 carbon) series determines whether the A and B ring fusion is trans (5a-series) or cis (5p- series).
HO'
The Influence o f Membrane Cholesterol onGABA a Currents page 31 Chapter 1: General Introduction
1.4.3 Sites of action for GAB A potentiation by neuroactive steroids
Since neuroactive steroids have a highly lipophilic property, they will accumulate in I the the plasma membrane and might interact withlGABÀA receptor by perturbing the membrane. However, this mechanism is unlikely because intracellular applications of neuroactive steroid, which should also accumulate in the inner leaflet of the plasma membrane, do not interfere with the effects of extracellularly applied steroid (Lambert et a l, 1990). Generally, it is accepted that the structure-activity requirements for neuroactive steroid activity point to specific binding sites on the GAB A A receptor protein (Simmonds, 1991; Lambert et a l, 1995; Zorumski et a l, 1996). However, the precise location(s) of the site(s) of action of neuroactive
steroids on the GABA a receptor is yet to be defined.
1.4.4 Neuroactive steroid antagonism of the GABAa receptor
Ithe In contrast to the positive modulatory neuroactive steroids which potentiatejresponse
to GAB A at the GABA a receptor, certain neuroactive steroids, such as pregnenolone sulphate and dehydroepiandrosterone sulphate, behave as noncompetitive antagonists of this receptor (Majewska & Schwartz, 1987; Majewska, 1992). The site of action of these sulphated steroids seems to be distinct from that of the positive modulatory neuroactive steroids (Park-Chung et a l, 1999). These steroids also act at other receptors. For example, they potentiate the effectiveness of glutamate action via Ithe NMD A receptor (Park-Chung et a l, 1997) and reduce voltage-activated calcium currents (ffrench-Mullen & Spence, 1991).
The Influence of Membrane Cholesterol onGABA a Currents page 32 Chapter 1: General Introduction
1.5 Effect of membrane lipids on the GABAa receptor
Some membrane lipids have been reported to affect certain properties of the GABA a receptor as revealed by benzodiazepine receptor binding studies. Phosphatidylserine facilitated the binding of [^H]flunitrazepam to the GABA a receptor and concomitantly inhibited the facilitation of [^H]flunitrazepam binding by GAB A and cartazolate in rat cerebellar synaptic membrane preparations (Hammond & Martin, 1987). To maintain modulatory characteristics in receptor binding experiments after solubilisation of the GABA a receptor by 3-[(3-cholamidopropyl)dimethylammonio]- 1-propane sulphonate (CHAPS), this procedure must be carried out in the presence of natural lipid extract and cholesterol hemisuccinate (Bristow & Martin, 1987). Moreover, cholesterol is found to be an essential requirement for the efficient reconstitution of this receptor complex into liposomes (Bristow & Martin, 1987). Cholesterol enrichment of rat synaptic membranes did not affect the affinity of the benzodiazepine site for flunitrazepam but the enhancement of [^HJflunitrazepam binding by pregnanolone and propofol was changed, the direction of change depending on the brain region (Bennett & Simmonds, 1996).
1.6 Aims and objectives
The evidence presented above gave strong encouragement to the notion that membrane cholesterol might affect the functional properties of the GABA a receptor. It was not possible to study all the variables that might be of interest so attention was focused on the quantitative pharmacological properties of a native population of receptors. Thus, published procedures to isolate neurones from rat hippocampus were further developed to permit enrichment and depletion of the membrane cholesterol content of these neurones whilst maintaining the viability required to
The Influence of Membrane Cholesterol on GABA^ Currents page 33 Chapter 1: General Introduction
measure GABA a currents using the whole-cell application of the patch clamp technique. Since the neuroactive steroids are structurally related to cholesterol, one particular hypothesis tested was that cholesterol might compete with the neuroactive steroids for their binding sites on the GABA a receptor protein and, thereby, selectively reduce their potency, compared with effects on other classes of modulatory drugs.
The Influence of Membrane Cholesterol on GABA^ Currents page 34 CHAPTER 2
MATERIALS AND METHODS
The Influence of Membrane Cholesterol on GABA^ Currents page 35 Chapter 2: Materials and Methods
2.1 Materials and Methods
2.1.1 Liposome preparation
Liposomes were prepared by the method of Bennett and Simmonds (1996) with minor modifications. Equal weights of phosphatidylcholine (PC) (99% PC from egg yolk or asolectin, crude phospholipid extracted from Soya bean) and cholesterol were dissolved in a small amount of chloroform. The chloroform was evaporated under a stream of nitrogen at room temperature to leave a thin film of lipids on the wall of the vessel. Physiological salt solution (PSS) containing (mM): NaCl 140, KCl 4.7, CaCl]
2.5, MgCl 2 1.2, Glucose 11, HEPES 10, adjusted to pH 7.4 with Tris-base was added in a volume of 1 ml for every 1.5 mg of cholesterol + 1.5 mg PC. The lipids were allowed to hydrate for 1 hour (hr) at room temperature. The lipids were then dispersed with a Branson Sonifier 250 (Branson Ultrasonics, USA) fitted with a half inch tip on 80% duty cycle under a stream of nitrogen, whilst incubating on an ice bath. Five periods of 4 min sonication were carried out at 75 W allowing the same time in between each sonication for cooling. After sonication the solution was centrifuged at 48400 xg (J2- 21M/E, BECKMAN, UK) for 20 min at 4°C and any pelleted material discarded.
Liposomes containing pure PC or asolectin only were prepared by the same procedure except PSS was added in a volume of 1 ml for every 3 mg PC.
2.1.2 Preparation of methyl-P-cyclodextrin solution and its cholesterol inclusion complex
Methyl-P-cyclodextrin was dissolved in PSS at a concentration of 100 mM and added to the neurone suspension to make concentrations of 5 or 10 mM.
The Influence of Membrane Cholesterol on GABA^ Currents page 36 Chapter 2: Materials and Methods
For preparing cholesterol-methyl-P-cyclodextrin complex, cholesterol was added to
30 mM methyl-P-cyclodextrin in PSS to make a concentration of 3 mM. The mixture was overlaid with nitrogen in an airtight glass bottle and stirred at room temperature (~20°C) until complete dissolution of the cholesterol, about 6 hr. The clear solution was filtered through a 0.22 pm Millipore filter (Millipore, UK). The cholesterol-methyl-P-cyclodextrin inclusion complex solution was overlaid with nitrogen and kept at -20°C.
2.1.3 Preparation of epicholesterol-methyl-P-cyclodextrin inclusion complex
Epicholesterol-methyl-p-cyclodextrin complex was prepared by the same procedure as cholesterol-methyl-P-cyclodextrin complex except the mixture was stirred until complete dissolution of the epicholesterol for about 24 hr.
2.1.4 Dissociation of hippocampal neurones
Hippocampal neurones were dissociated from male Wistar rats aged 10-16 days by a method modified from Kaneda et al. (1988) and Kara et al. (1993). Rats were killed by cervical dislocation and decapitated. Brains were rapidly removed and sliced into 400 pm thick sections with a Vibroslice (Campden Instruments, UK). After 30-60 min preincubation in oxygen-saturated PSS, the slices were enzymatically treated in PSS containing 0.03% (w/v) protease type XTV (pronase), followed by 0.03% (w/v) protease type X (thermolysin) for 20 min each at 31°C. After the enzyme treatment, the slices were washed three times with PSS.
The hippocampal tissue was isolated and the neurones were dissociated by gentle trituration with fire-polished glass pipettes. The neurone suspension was allowed to stand for 1 0 min before the supernatant containing dissociated neurones was taken off.
The Influence of Membrane Cholesterol onGABA a Currents page 37 Chapter 2: Materials and Methods
leaving only 0.3 - 0.4 ml containing big lumps. The suspension was then centrifuged at 175 xg (Centaur 2E, MSE, UK) for 3 min. The loose pellet of neurones was resuspended in fresh PSS and layered onto a 5% (w/v) solution of bovine serum albumin (BSA) in PSS for further centrifugation at 175 xg for 4 min to separate a loose pellet of neurones from suspended cell debris.
The neurones were suitable for electrophysiological recording after being allowed to adhere to the base of a recording chamber for 30-40 min.
2.1.5 Manipulation of membrane cholesterol using liposomes
1) Cholesterol enrichment
After purification of the dissociated hippocampal neurones, one half of the neurone suspension was subjected to cholesterol enrichment using the liposome incubation method modified from Bennett and Simmonds (1996). The neurones were incubated in oxygen-saturated PSS containing 1.0% (w/v) BSA and cholesterol-phosphatidylcholine or cholesterol-asolectin liposomes at a final cholesterol concentration of 0.52 mM (0.20 mg.ml'^) at 31°C for 60 or 120 min. The cholesterol transfer was terminated by centrifugation at 175 xg for 3 min and two washes of the neurones with PSS. For the control group (untreated neurones) the other half of the neurone suspension was subjected to the same procedure but without liposomes.
The Influence of Membrane Cholesterol on GABA^ Currents page 38 Chapter 2: Materials and Methods
2) Cholesterol depletion
Samples of the original neuronal suspensions were incubated with liposomes containing PC or asolectin only under the same conditions as described in cholesterol enrichment for 30, 60 or 120 min.
2.1.6 Manipulation of membrane cholesterol using methyl-P-cyclodextrin or its cholesterol inclusion complex.
1) Cholesterol enrichment
One half of the neurone suspension was incubated in oxygen-saturated PSS containing cholesterol-methyl-p-cyclodextrin complex at a final concentration of 0.15 or 0.3 mM cholesterol at 31°C for 5-30 min depending on the degree of enrichment required. The cholesterol transfer was terminated by centrifugation at 340 xg for 3 min and two washes of the neurones with PSS. For the control group (untreated neurones) the other half of the neurone suspension was subjected to the same procedure but without cholesterol-methyl-P-cyclodextiin complex.
2) Cholesterol depletion
Samples of the original neuronal suspensions were incubated with 5 or 10
mM methyl-P-cyclodextrin solution under the same conditions as described in cholesterol enrichment.
The Influence of Membrane Cholesterol on GABA^ Currents page 39 Chapter 2: Materials and Methods
3) Restoration of cholesterol following depletion
The neurones were incubated in oxygen-saturated PSS containing 10 mM methyl-P-cyclodextrin at 31°C for 20 min. The cholesterol transfer was terminated by centrifugation at 340 xg for 3 min. The pellet was resuspended and the neurones were incubated in oxygen-saturated PSS containing cholesterol-methyl-P-cyclodextrin complex at a final concentration of 0.1 mM cholesterol at 31°C for 1-10 min. The cholesterol transfer was terminated by centrifugation at 340 xg for 3 min and two washes of the neurones with PSS.
2.1.7 Incubation of the neurones with epicholesterol using epicholesterol- methyl-P-cyclodextrin inclusion complex.
1) Incubation of control neurones with epicholesterol
The neurone suspension was incubated in oxygen-saturated PSS containing epicholesterol-methyl-p-cyclodextrin complex at a final concentration of 0.15 mM epicholesterol at 31°C for 5 and 20 min. The transfer of epicholesterol was terminated by centrifugation at 340 xg for 3 min and two washes of the neurones with PSS.
2) Incubation of cholesterol-depleted neurones with epicholesterol
The neurones were incubated in oxygen-saturated PSS containing 10 mM
methyl-P-cyclodextrin at 31°C for 20 min. The cholesterol transfer was terminated by centrifugation at 340 xg for 3 min. The pellet was resuspended and the neurones were, then, incubated in oxygen-saturated
The Influence of Membrane Cholesterol on GABA^ Currents page 40 Chapter 2: Materials and Methods
PSS containing epicholesterol-methyl-P-cyclodextrin complex at a final concentration of 0.1 mM epicholesterol at 31°C for 5-20 min. The transfer of epicholesterol was terminated by centrifugation at 340 xg for 3 min and two washes of the neurones with PSS.
2.1.8 Cholesterol and protein assays of hippocampal neurones
After plating samples of the neurone suspension for electrophysiological recording, the remaining neurones were frozen for later assay for cholesterol and protein. The neurones were homogenized (Ultra Turrax Homogenizer, Germany) in ice cold wash buffer containing (mM): Tris-base 5, EDTA 1, pH 7.4 at 4°C, centrifuged at 48,400 xg (J2-21M/E, BECKMAN, UK), and then resuspended in ice cold wash buffer.
Cholesterol assay was carried out on the suspensions using the Sigma cholesterol diagnostic kit that measured total cholesterol enzymatically and was modified from the method of Allain et a l (1974). Cholesterol esters were hydrolysed by cholesterol esterase to cholesterol. The cholesterol produced by hydrolysis and free cholesterol were oxidised by cholesterol oxidase to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide was then coupled with the chromogen, 4-aminoantipyrine and p-hydroxybenzenesulfonate in the presence of peroxidase to yield a quinoneimine dye which had an absorbance maximum of 500 nm. The standard curve for cholesterol assay is shown in figure 2.1. Cholesterol calibrator (Sigma, USA) was used as standard cholesterol. The absorbances measured from all experiments were in the range of 0.004-0.020.
Protein assay was carried out on the suspensions using the BioRad Protein Assay. This assay is based on the shift of the absorbance maximum from 465 nm to 595 nm of Coomassie brilliant blue G-250 dye in response to various concentration of protein (Bradford, 1976).
The Influence o f Membrane Cholesterol on GABA^ Currents page 41 Chapter 2: Materials and Methods
Figure 2.1 Standard curve of cholesterol measured by cholesterol diagnostic kit (Sigma, USA). Cholesterol calibrator (Sigma, USA) was used as standard cholesterol. All data points are the means of triplicates.
0.04-1
0 . 03 - 0 .0 1 - 0.00 0.00 0.05 0.10 0.15 0.20 0.25 Cholesterol conccentration (mg/ml) The Influence of Membrane Cholesterol on GABA^ Currents page 42 Chapter 2: Materials and Methods 2.1.9 Electrophysiological measurements The effects of membrane cholesterol on GABAa receptors were investigated in acutely dissociated hippocampal neurones using the whole-cell application of the patch clamp technique (Hamil et a l, 1981). Whole-cell membrane currents were recorded from the somata of the dissociated hippocampal neurones voltage-clamped at -20 mV (holding potential). Patch pipettes were prepared from thin wall borosilicate glass capillaries (Clark Electromedical Instrument, UK) on a two-stage vertical puller (KOPF, USA). The patch-pipette was filled with the intrapipette solution containing (mM): CsCl 140, MgCl 2 4, Naz ATP 4, EOT A 11, CaCli 1, and HEPES 10, adjusted to pH 7.2 with Tris- base. The resistance between the patch-pipette and a reference electrode in the external solution (PSS) ranged from 3-5 MQ. Membrane current was measured with a patch clamp amplifier (EPC-7, List-Electronic, Germany) and was monitored simultaneously on an oscilloscope (GOULD, UK) and a pen recorder (GOULD, France). During recording, neurones were superfused with PSS at a rate of 1.5-2.0 ml/min. All experiments were performed at room temperature (~20°C). 2.1.10 Drugs and Chemicals GAB A was dissolved in PSS. Cholesterol for acute application was dissolved in acetone at a concentration of 40 mM, and mixed with dimethyl sulfoxide (DMSO) to achieve a concentration of 8 mM and then diluted to 1 and 4 pM in PSS. The final concentration of acetone was less than 0.0025 and 0.01% (v/v), respectively. Pregnanolone, allopregnanolone, alphaxalone, propofol and bicuculline methochloiide were dissolved in DMSO at a concentration 10 mM. Flunitrazepam and picrotoxinin were dissolved in DMSO at a concentration 20 mM. Pregnenolone sulphate and pentobarbital sodium were dissolved in DMSO at concentration 40 mM. All of drug stock solutions were then diluted to appropriate concentrations in PSS. The The Influence of Membrane Cholesterol onGABA a Currents page 43 Chapter 2: Materials and Methods concentration of DMSO was less than 0.1% (v/v) throughout and it was confirmed that this concentration had no effect on its own in separate control experiments. Rapid application of drugs was performed with the “U-tube” minor modification from Fenwick et a l, 1982. A U-shaped glass capillary of diameter -150 |xm with a small circular hole (-50 pm) at the tip was placed about 300 pm from the neurone to be recorded. One end of the U-tube was connected to a reservoir of external solution or drug solution held about 20 cm above the level of the U-tube tip. The other end was connected to a vacuum pump (HY-FLO, Medcalf Bros., UK) which sucked solution from the reservoir through the U-tube and also drew external solution into the tube through the hole at its tip. To reverse the flow through the hole of the U-tube, the line to the vacuum pump was occluded for 3 seconds (sec) to allow gravity to drive the solution from the tube and superfuse the recorded neurone. Increasing concentrations of G ABA, 0.1-1000 pM, in the absence of modulatory drug were applied to a neurone first at 1 min interval, and then increasing concentrations of GAB A in the presence of drug in the same pipette were applied subsequently to the same neurone. In case of bicuculline and picrotoxinin, increasing concentrations of 0.3-10,000 pM GAB A in the presence of the antagonists were applied subsequently to the neurones. All drugs and chemicals were purchased from Sigma (USA) with some exceptions. Alphaxalone was a gift from Glaxo Group Research (Greenford, UK). Methyl-p- cyclodextrin was purchased from Aldrich (UK). Epicholesterol was purchased from Steraloids (USA). BioRad Protein Assay was purchased from BIO-RAD (Germany). CsCl, MgCE, CaClz and acetone were purchased from BDH (Poole, UK). The Influence of Membrane Cholesterol onGABA a Currents page 44 Chapter 2: Materials and Methods 2.1.11 Data Analysis Data are expressed as mean + standard error of the mean (S.E.M.). To combine data from different neurones, all G ABA responses were expressed as percentages of the maximum response of the same neurone. For concentration-response analysis, data were plotted using GraphPad FRISNT^ version 3.0 (GraphPad Software, USA) and fitted with a logistic equation in the form: I = Imin + (Inm-Imin)/(l+(EC5o/[X])”) where I is GABAa current, Imin and Imax are the minimal and the maximal currents recorded in a given neurone, respectively, EC 50 is the concentration of G ABA eliciting 50% of the maximal current, [X] is the G ABA concentration, and H is the Hill coefficient. For statistical analysis of the effect of membrane cholesterol on the potencies of the modulators, a ratio of EC 50 values for GAB A in the presence and absence of modulator was determined from each experiment. Significance was tested by Student’s unpaired r-test or ANOVA where applicable, and a P value of less than 0.05 was considered to be significant. The Influence of Membrane Cholesterol on GABA^ Currents page 45 Chapter 2: Materials and Methods 2.2 Discussion To study the effects of membrane cholesterol alterations on the GABA a receptors, two carriers of cholesterol, liposomes and methyl-P-cyclodextrin, were used to transfer cholesterol from or to the plasma membrane of acutely dissociated hippocampal neurones. Liposomes are simply vesicles in which an aqueous compartment is entirely enclosed by a membrane composed of lipid molecules (usually phopholipids). They can be constructed to entrap many chemicals and drugs both within their aqueous compartment and within the membrane (New, 1990). In the present study, liposomes were constructed to entrap cholesterol and acted as cholesterol donors to transfer cholesterol to dissociated neurones in the cholesterol enrichment procedure. The liposomes prepared without cholesterol were also used as acceptors of cholesterol transferred from the neurones in the cholesterol depletion procedure. In the early experiments, asolectin was used to prepare liposomes, based on the method described by Bennett & Simmonds (1996). Asolectin is a crude soya bean lipid preparation consisting of PC and other phospholipids such as phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin as its main constituents. There was some concern that some of these lipids, as well as cholesterol, might enter the neuronal plasma membranes with functional consequences. In later experiments, therefore, asolectin was replaced by a pure PC preparation from egg yolk. It will become apparent, in subsequent Chapters, that this substitution made little difference to the results obtained. Later still, the use of methyl-P-cyclodextrin as described by Gimpl et al. (1997) came to our attention as both a technically simpler method than the use of liposomes and one which is capable of more substantial depletions of membrane cholesterol than could be achieved with liposomes. Cyclodextrins are water-soluble cyclic oligomers The Influence of Membrane Cholesterol onGABA a Currents page 46 Chapter 2: Materials and Methods of glucose with a hydrophobic cavity capable of enhancing the solubility of nonpolar substances by incorporating them into their hydrophobic cavity and forming inclusion complexes (Pitha et al., 1988). Because of the ability to increase solubility of lipophilic drugs, cyclodextrins or their derivatives have been used extensively as drug delivery vehicles (Pitha et a l, 1988). The three different cyclodextrins (a-, p- and y- cyclodextrins) have been used to alter the lipid composition of erythrocytes, and P~ cyclodextrin was found to selectively extract cholesterol from plasma membrane in preference to other membrane lipids (Ohtani et al, 1989). The cyclodextiin that is best suited to form stable complexes with cholesterol is methyl-p-cyclodextrin (Klein et a l, 1995). Methyl-p-cyclodextrin is also the most effective and selective substance, compared to other P-cyclodextrins (P-cyclodextrin and 2-hydroxypropyl-P- cyclodextiin), to deplete cholesterol from cultured cells (Kilsdonk et a l, 1995). The incubation protocols with methyl-p-cyclodextrin described in this Chapter were developed to provide the range of graded cholesterol enrichments and depletions described in Chapter 3. For the experiments in which epicholesterol was included with methyl-P- cyclodextrin, there was no assay method for the epicholesterol available to us and reliance has, therefore, been placed on the work of Gimpl et a l (1997) which shows that epicholesterol incorporates into plasma membranes just as readily as cholesterol from the methyl-p-cyclodextrin carrier system. The method employed to dissociate hippocampal neurones in this study was slightly modified from Kaneda et a l (1988). They found that the useful proteases for neuronal dissociation were pronase and thermolysin. The advantages of this method are that intact single neurones can be routinely dissociated, all dissociated neurones preserve their morphological structure and the electrical and chemical properties of the dissociated neurones are well maintained. From the preliminary study, it was The Influence of Membrane Cholesterol on GABA^ Currents page 47 Chapter 2: Materials and Methods found that the best ages of rats for neurone dissociation were between 10-16 days. Dissociation from this group of rats had a higher percentage of viable neurones and less cell debris than from the rats aged 3-4 weeks. Due to the limitation of protein and cholesterol assays, rats aged less than 10 days did not have enough tissues for the assays. After dissociation, neurones were purified by gravity sedimentation and centrifugation through 5% (w/v) BSA to eliminate big lumps and cell debris, which would otherwise have contributed to the assays of neuronal cholesterol and protein. Centrifuging the neurones through BSA solution was also important to ensure the long term survival (Boss et a l, 1987). Although most of the cells after purification were neurones, some glial cells were also found in this preparation. G ABA and drugs were applied rapidly to the neurones using the U-tube method (Fenwick et a l, 1982). By this technique, the external solution surrounding a neurone could be exchanged within 50 ms, so that the accurate peak value of the response could be obtained before substantial desensitization develops. GAB A in the absence or in the presence of drug was briefly applied to the neurone for 3 sec at 1 min intervals. Immediately following the application, the drugs in the vicinity of the recorded neurone would have been washed away in a few seconds by the continuous perfusion of the recording chamber. Thus, with repeated applications of GAB A and a modulatory drug, the neurone was exposed to the drug for about 5 sec every 1 min. For lipid-soluble drugs that may need to accumulate in the neuronal membrane to exert their effects, equilibrium was unlikely to have been reached until after the second or third applications. Since the initial applications always contained low concentrations of G ABA, any lack of equilibrium of the modulatory drug would have been confined to the bottom of the GAB A dose-response curve and should have had little consequence for the estimations of G ABA EC 5 0 . The alternative approach of superfusing the modulatory drug through the recording chamber prior to local application of G ABA was not used. This was to avoid The Influence of Membrane Cholesterol on GABA^ Currents page 48 Chapter 2: Materials and Methods exposure of other neurones in the chamber and, thereby, allow them to be used subsequently for analogous experiments. The Influence of Membrane Cholesterol onGABA a Currents page 49 CHAPTER 3 MANIPULATION OF MEMBRANE CHOLESTEROL The Influence of Membrane Cholesterol onGABA a Currents page 50 Chapter 3: Manipulation of Membrane Cholesterol 3.1 Introduction Several methods have been developed to alter the cholesterol content of the plasma membranes of cells, whether in the form of membrane fragments prepared from cells (Whetton et ah, 1983; Bennett & Simmonds, 1996) or acutely isolated whole cells (Sooksawate & Simmonds, 1998) or cultured cells (Yancey et al., 1996; Gimpl et al., 1997). These methods included the use of non-specific lipid transfer proteins (Castuma, & Brenner, 1986), the preparation of highly diluted aqueous cholesterol solutions (Crews et al., 1983), the use of cholesterol oxidase (Gimpl et al., 1997), incubation with liposomes (Whetton et a l, 1983; Bennett & Simmonds, 1996), and the use of cyclodextrin and its cholesterol inclusion complex (Kilsdonk et al., 1995; Gimpl et al., 1997). Incubations with liposomes or with cyclodextrin and its cholesterol inclusion complex appear to be the most favoured methods and were adopted in the present study. The objective was to explore how far membrane cholesterol could be enriched or depleted to a stable level, compatible with neuronal viability and ability to make satisfactory whole cell patch electrode recordings. The Influence of Membrane Cholesterol onGABA a Currents page 51 Chapter 3: Manipulation of Membrane Cholesterol 3.2 Experimental protocols and results 3.2.1 The viability of acutely dissociated hippocampal neurones After dissociation, about 90% of hippocampal neurones were viable (trypan blue exclusion). The neurones remaining viable after 1 hr incubation with and without liposomes were 70-80% of liposome-incubated (enriched or depleted) and 80-90% of control neurones. After incubation with 10 mM methyl-|3-cyclodextrin or 0.15 mM cholesterol-methyl-(3-cyclodextrin inclusion complex for less than 30 min, the neurones remaining viable were 70-90%. Most of the neurones after incubation with liposomes or methyl-(3-cyclodextrin had resorbed their processes and were rounded up. Less than 50% of the neurones were still viable after plating into a recording chamber for 4 hr. Photomicrographs of control, liposome-incubated and methyl-p- cyclodextrin-incubated neurones are shown in figure 3.1. 3.2.2 Alterations of membrane cholesterol content by liposomes 1) Asolectin liposomes After incubation with cholesterol-asolectin liposomes for 60 min, cholesterol content of hippocampal neurones could be enriched to 127±1.67% of control (n=10). In contrast, after incubation with asolectin liposomes (without cholesterol) for 60 min, cholesterol content of the neurones could be depleted to 82.1±0.61% of control (n=5). The absolute cholesterol levels of these neurones are shown in table 3.1. The neurones subjected to those incubations remained in a satisfactory condition for whole-cell patch clamp recordings. The Influence o f Membrane Cholesterol onGABA a Currents page 52 Chapter 3: Manipulation of Membrane Cholesterol Figure 3.1 Photomicrographs of acutely dissociated hippocampal neurones from 10-16 days old Wistar rat brains before and after membrane cholesterol alterations. The neurones were incubated at 31°C with phosphatidylcholine (PC) liposomes or cholesterol-PC liposomes for 1 hr, and methyl-P-cyclodextrin or its cholesterol inclusion complex for 20 min. (A) control neurones, (B) cholesterol-depleted (82% of control) neurones (PC-liposomes), (C) enriched (127%) neurones (cholesterol-PC liposomes), (D) depleted (56%) neurones (methyl-P-cyclodextrin), (E) enriched (182%) neurones (cholesterol-methyl-P-cyclodextrin). Scale bar, 25 pm. D E The Influence of Membrane Cholesterol on GABA^ Currents page 53 Chapter 3: Manipulation of Membrane Cholesterol Table 3.1 Alterations of membrane cholesterol by liposomes in acutely dissociated rat hippocampal neurones. Membrane cholesterol was manipulated by incubation at 3I°C for 30-120 min with liposomes prepared from asolectin or pure egg yolk phosphatidylcholine (PC) without (depletion) or with cholesterol (Ch) (enrichment) (for details of method see Chapter 2). Cholesterol content of neurones (pmole.mg protein *) Incubation Depletion Enrichment tim e (m in) A solectin Pure PC Ch-asolectin Ch-pure PC liposom es lip osom es lip osom es liposom es Control 0.309±0.009 (5) 0.319+0.009 (9) 0.373±0.009 (10) 0.299±0.012 (6) 30 - 0.298+0.014* (3) -- 60 0.254±0.009*** (5) 0.270±0.012*** (3) 0.473+0.012*** (10) 0.363±0.017** (3) 120 - 0.254±0.012*** (3) - 0.390±0.030** (3) Numbers of experiments are shown in parentheses. * P<0.05 (from control, Student’s r-test), ** P<0.001, *** P<0.0001 2) Pure PC liposomes The same results as obtained from using azolectin liposomes could also be found by using liposomes prepared from pure egg yolk PC. Cholesterol content of the neurones could be enriched to 122±1.71% and 130.0±1.50% of control neurones containing 0.299±0.012 (n= 6 ) |xmoles cholesterol.mg protein'^ after incubation with cholesterol-PC liposomes for 60 and 120 min, respectively (Figure 3.2 and Table 3.1). The Influence of Membrane Cholesterol onGABA a Currents page 54 Chapter 3: Manipulation of Membrane Cholesterol Figure 3.2 Alterations of membrane cholesterol by liposomes in acutely dissociated rat hippocampal neurones. Membrane cholesterol was manipulated by incubation at 31°C for 30-120 min with pure phosphatidylcholine liposomes without (depletion) or with cholesterol (enrichment) (for details of method see Chapter 2) (n=3). 150i * P<0.01 (from control. Student's t-test) ** P<0.()05 c uo 125- If 100 - I 75- D epletion Enrichment — r ~ — i— 30 60 90 120 Incubation time (min) The Influence of Membrane Cholesterol on GABA^ Currents page 55 Chapter 3: Manipulation of Membrane Cholesterol In contrast, after incubation with pure PC liposomes (without cholesterol) for 30, 60 and 120 min, cholesterol content of the neurones could be depleted to 97.6±0.99%, 83.6±0.57% and 77.7±L09% of control neurones, respectively, containing 0.319±0.009 (n=9) jxmoles cholesterol.mg protein'^ (Figure 3.2 and Table 3.1). After incubation with liposomes (with or without cholesterol) for 1 hr, the success rate for whole-cell patch clamp recording in the neurones was satisfactory. However, it was quite difficult to make a stable whole cell recording in the neurones after 2 hr incubation with the liposomes. 3.2.3 Alterations of membrane cholesterol content by methyl-P-cyclodextrin and its cholesterol inclusion complex Greater alterations of membrane cholesterol in dissociated hippocampal neurones were obtained by incubation with methyl-P-cyclodextrin and its cholesterol inclusion complex than by incubation with liposomes. After incubation with cholesterol- methyl-P-cyclodextrin complex at a final concentration of 0.15 or 0.3 mM cholesterol for 5-30 min, cholesterol content of the neurones could be enriched within the range 136±1.97% to 191±2.16% of control neurones containing 0.319±0.009 (n=12) pmoles cholesterol.mg protein'^ or 149±4.25% to 235±5.40% of control neurones containing 0.331±0.011 (n=7) pmoles cholesterol.mg protein'\ respectively (Figure 3.3 and Table 3.2). The Influence of Membrane Cholesterol on GABA^ Currents page 56 Chapter 3: Manipulation of Membrane Cholesterol Figure 3.3 Alterations of membrane cholesterol by methyl-p-cyclodextrin in acutely dissociated rat hippocampal neurones. Membrane cholesterol was manipulated by incubation at 31°C for 5-30 min with 5 and 10 mM methyl-P- cyclodextrin (MPC) (depletion) or cholesterol-methyl-p-cyclodextrin complex (Ch- MPC) at a final concentration of 0.15 and 0.30 mM cholesterol (enrichment) (for details of method see Chapter 2) (n=3-6). 250n * P<0.05 (from control. Student's /-test) ** /^<0.0i *** P<0.00! ** 200 - I s ^ 2 150- 100 - 50-1 — □ r T TT 10 20 30 Incubation time (min) Depletion (5 mM M[3C) —•— Eniichment (0.15 mM Ch-MpC) Depletion (10 mM M(3C) —■— Enrichment (0.30 mM Ch-MPC) The Influence of Membrane Cholesterol on GABA^ Currents page 57 Chapter 3: Manipulation of Membrane Cholesterol Table 3.2 Alterations of membrane cholesterol by methyl-p-cyclodextrin in acutely dissociated rat hippocampal neurones. Membrane cholesterol was manipulated by incubation at 31°C for 5-30 min with 5 and 10 mM methyl-p-cyclodextrin (MpC) (depletion) or cholesterol-methyl-P-cyclodextrin complex (Ch-MpC) at a final concentration of 0.15 and 0.30 mM cholesterol (enrichment) (for details of method see Chapter 2). Cholesterol content of neurones (pmole.mg protein"') Incubation Depletion/Mpc (mM) Enrichment/Ch-MpC (mM) tim e (m in) 5 10 0.15 0.3 0 Control 0.325±0.010 (7) 0.30910.006 (14) 0.31910.009 (12) 0.33110.011 (7) 5 0.288±0.010* (3) 0.22510.017** (3) 0.42310.011** (3) 0.49710.031** (3) 10 0.26810.010** (3) 0.21810.005** (4) 0.51310.019** (4) 0.61510.056** (3) 20 0.23110.018** (3) 0.17510.010** (6) 0.56010.032** (5) 0.66110.049** (3) 30 0.20310.009** (3) 0.16610.012** (3) 0.63610.046** (3) 0.80910.029** (3) Numbers of experiments are shown in parentheses. * f <0.001 (from control, Student’s f-test), ** f <0.0001 In contrast, after incubation with 5 or 10 mM methyl-P-cyclodextrin for 5-30 min, cholesterol content of the neurones could be depleted within the range 89.2±1.55% to 61.9±1.46% of control neurones containing 0.325±0.010 (n=7) pmoles cholesterol.mg protein'^ or 72.0±1.19% to 53.9±2.53% of control neurones containing 0.309±0.006 (n=14) pmoles cholesterol.mg protein'\ respectively (Figure 3.3 and Table 3.2). Cholesterol enrichment by incubation with 0.15 mM cholesterol-methyl-p- cyclodextrin complex for up to 2 0 min was compatible with satisfactory whole-cell The Influence of Membrane Cholesterol on GABA^ Currents page 58 Chapter 3: Manipulation of Membrane Cholesterol patch clamp recordings. However, longer incubation times or an increase in the concentration of cholesterol in the methyl-p-cyclodextrin complex to 0.3 mM made successful and stable whole cell recordings more difficult to obtain. In the case of cholesterol depletion, satisfactory whole cell recordings could still be achieved after incubation with 5 or 10 mM methyl-P-cyclodextrin for up to 30 min. However, the depletion had reached its maximum (around 50% of control) after incubation for 20 min with 10 mM methyl-P-cyclodextrin. 3.2.4 Restoration of membrane cholesterol following depletion After cholesterol depletion (56% of control) by incubation with 10 mM methyl-p- cyclodextrin for 2 0 min, the membrane cholesterol levels could be restored to 79.8±4.03% (n=3), 89.2±3.07% (n=3) and 103±3.50% of control (n=3) by incubation with cholesterol-methyl-p-cyclodextrin complex at a final concentration of 0.1 mM cholesterol for 1, 5 and 10 min, respectively (Figure 3.4). The Influence of Membrane Cholesterol on GABA^ Currents page 59 Chapter 3: Manipulation of Membrane Cholesterol Figure 3.4 Restoration of membrane cholesterol following depletion. Membrane cholesterol was depleted by incubation at 31®C for 20 min with 10 mM methyl-f- cyclodextrin (MpC) and subsequently restored by incubation at 31°C for 1-10 min with cholesterol-methyl-p-cyclodextrin complex at a final concentration of 0.1 mM cholesterol (for details of method see Chapter 2) (n=3-6). 1 4 0 - Dcplction Repletion Incubated with Incubated with 10 mM MpC 0.1 mM Ch-MBC 20 Incubation time (min) * f<0.05 (from control. Student's t-test) ** P<0.005, *** P<0.0001 nonsignificant difference The Influence of Membrane Cholesterol on GABA^ Currents page 60 Chapter 3: Manipulation of Membrane Cholesterol 3.3 Discussion The methods employed to alter membrane cholesterol content of acutely dissociated rat hippocampal neurones in this study were (1) the incubation with cholesterol-PC liposomes or liposomes containing PC only in the presence of 1% (w/v) BSA, and (2) the incubation with methyl-P-cyclodextrin or its cholesterol inclusion complex. From the fact that the plasma membrane of a cell contains more than 90% of the cholesterol present within that cell (Yeagle, 1993), we have presumed that the extra cholesterol accumulated during the cholesterol enrichment procedure was located predominantly in the plasma membrane. For the cholesterol depletion procedure, we have also presumed that cholesterol was transferred from the plasma membrane to the acceptors of cholesterol, liposomes or methyl-P-cyclodextrin. Incubation with liposomes prepared from asolectin, a crude lipid extract from soya bean, or pure egg yolk PC about equally altered membrane cholesterol of acutely dissociated hippocampal neurones (Table 3.1). The enrichment with cholesterol achieved in these neurones was substantially less than the doubling routinely observed in synaptosomal membrane fragments (Bennett & Simmonds, 1996; Schlepper et a l, 1998). In part this may have been due to the more cautious application of the enrichment procedure to minimise loss of viable neurones; thus, the neurones were incubated with liposomes for only 1-2 hr rather than 4 hr and the neurones were maintained in PSS during the enrichment procedure rather than the optimal low ionic strength medium (Crain & Ziversmit, 1980) used with the membrane fragments. However, the liposome incubation method was less effective than incubation with methyl-p-cyclodextrin or its cholesterol inclusion complex in the alteration of membrane cholesterol levels in these neurones (Figure 3.2-3.3, Table 3.1-3.2). This result was in agreement with Kilsdonk et al. (1995) who used mouse L-cell The Influence of Membrane Cholesterol on GABA^ Currents page 61 Chapter 3: Manipulation of Membrane Cholesterol fibroblasts and GM3486A human fibroblasts in their experiments. A further advantage of using methyl-P-cyclodextrin and its cholesterol inclusion complex is that the membrane cholesterol of membrane fragments and cultured cells can be reversibly changed (Gimpl et al., 1997). This was exploited in the present study where initial cholesterol depletion by incubation with methyl-P-cyclodextrin was followed by restoration back to control level by incubation with cholesterol-methyl- p-cyclodextrin complex (Figure 3.4). Such a procedure allowed discrimination between cholesterol-dependent changes in neuronal properties and changes due to some effects of methyl-P-cyclodextrin. The changes in membrane cholesterol reported here with methyl-P-cyclodextrin as a cholesterol carrier were sufficient for the purposes of this study but more extreme changes were found to be incompatible with satisfactory electrophysiological recordings and with neuronal viability. Since membrane cholesterol was measured immediately after the incubation procedures, it raised the question whether or not these levels fairly reflected the condition of the single neurones at the time of recording. The very small amount of tissue plated out for whole-cell patch clamp recordings was insufficient for a cholesterol assay to be performed at the end of the experiment. However, the increases of GAB A EC 50 caused by both cholesterol depletion and enrichment were highly reproducible (see Chapter 4 for details) despite their being obtained from neurones over a wide time range, from 40 min after plating to 4-6 hr after plating. This suggests that any change, in membrane cholesterol in either depleted or enriched neurones over this period was not significant. Despite extensive investigation, the principal mechanisms by which cholesterol is transferred between donor and acceptor membranes are not fully established (Bittman, 1993). Although strong evidence has been presented in favour of the aqueous diffusion pathway for cholesterol exchange (Bittman, 1993), the collisional complex pathway has been postulated in some systems (Miitsch et a l, 1986; Steck et The Influence of Membrane Cholesterol onGABA a Currents page 62 Chapter 3: Manipulation of Membrane Cholesterol a l, 1988). The aqueous diffusion mechanism required the desorption of cholesterol from the donor into the aqueous phase followed by the incorporation of the desorbed cholesterol molecules into the acceptor particles. The rate-limiting step of this mechanism is the release of free cholesterol molecule out of the donor into the aqueous phase (Bittman, 1993). Since the size of cyclodextrin (diameter -15 Â) is smaller than liposomes (127+1.10 nm, 3 experiments, measured by photon correlation spectroscopy, AutoSizer 2C, Malvern Instrument, UK) used in this study, cyclodextrin can access the region very close to the cell plasma membrane whereas liposomes may be restricted from this site (Davidson et a l, 1995). Thus, cholesterol molecules can transfer directly between the plasma membrane of the cells and the hydrophobic core of a cyclodextrin molecule accumulated near the membrane surface without necessity of desorbing completely into the aqueous medium (Yancey et a l, 1996). This may explain the much higher transfer of cholesterol observed with methyl-p-cyclodextrin when compared with liposomes. Also, it should be noted that inclusion of protein in the aqueous medium, presumably as a cholesterol carrier, is necessary for transfer between liposomes and neuronal membranes (Bennett & Simmonds, 1996) but is not required when methyl-p-cyclodextrin is used. The Influence of Membrane Cholesterol on GABA^ Currents page 63 CHAPTER 4 EFFECT OF MEMBRANE CHOLESTEROL ON THE SENSITIVITY OF GABAa r e c e p t o r s TO GABA The Influence of Membrane Cholesterol onGABA a Currents page 64 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA 4.1 Introduction In every experiment in which the potency of GABA potentiators or antagonists was to be studied, a control concentration-response curve to GABA was determined. The accumulated control data are presented in this chapter and analysed to discern any effects of cholesterol enrichment or depletion on the GABA a receptor sensitivity to GABA. Additional experiments are also included to check for possible effects of methyl-P- cyclodextrin, independent of the effects of altered cholesterol levels, and also to check for possible effects of cholesterol leaching out of the membrane to interact with the extracellular surface of theGABA a receptor. Current-voltage relationships of the GABA response were also determined to check for cholesterol-induced changes in voltage dependency. The Influence of Membrane Cholesterol onGABA a Currents page 65 ______Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivity of aGABA Receptors to GABA 4.2 Experimental protocols and results 4.2.1 Effect of membrane cholesterol on the GABA EC 50 1) Membrane cholesterol alterations by incubation with liposomes (see section 3.2.2, Chapter 3) The currents induced by increasing the concentration of GABA on control, cholesterol-depleted and enriched neurones, by incubation with pure PC liposomes or cholesterol-PC liposomes, are shown in figure 4.1. The GABA log.concentration-response curves of these neurones are shown in figure 4.2. At these low level changes of membrane cholesterol, both depletion (78% of control by incubation with pure PC liposomes for 2 hr) and enrichment (130% of control by incubation with cholesterol-PC liposomes for 2 hr) were associated with significant (P< 0 .0 0 0 1 ) increases in the E C 5 0 values for the GABA log.concentration-response relationship (Figure 4.3) with no change in Hill slope (Figure 4.2). In control, depleted and enriched neurones, the GABA EC 50 values were 4.96±0.16 pM (n=9), 10.72±0.53 pM (n=9) and 7.3810.24 pM (n=12), respectively. As shown in pure PC liposome experiments, after incubation of the neurones with asolectin liposomes (with or without cholesterol) for 1 hr, cholesterol depletion (82% of control) and enrichment (127% of control) also increased the GABA E C 5 0 values to 7.8110.16 pM (P<0.0001, n=9) and 5.8810.17 pM (P<0.01, n=16), respectively, compared with 4.9610.16 pM (n=9) in control neurones. The Influence of Membrane Cholesterol onGABA a Currents page 66 Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivity ofGABA^ Receptors to GABA Figure 4.1 The effect of membrane cholesterol alterations by liposomes on GABA a currents in acutely dissociated hippocampal neurones. Membrane cholesterol was manipulated by incubation at 31°C with pure PC liposomes (cholesterol depletion) or 0.52 mM cholesterol-PC liposomes (enrichment) for 1-2 hr. (A) Control neurone, (B) depleted neurone (78% of control), (C) enriched neurone (130% of control). G A B A (fiM ) 0.1 0.3 1 3 10 30 100 300 1000 i r 1 I' 20 s A. Control neurone GABA (^M ) 0.1 0.3 1 3 10 30 100 300 1000 ^ ^ ^ j - p > ■ 20 s B. Depleted neurone GABA (^iM) 0.1 0.3 1 3 10 30 100 300 1000 1 ■ ' t ...... L I" 20 s C. Enriched neurone The Influence of Membrane Cholesterol on GABA^ Currents page 67 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA ofa Receptors to GABA Figure 4.2 The effect of membrane cholesterol alterations by liposomes on GABA concentration-response relationship. Membrane cholesterol was manipulated by incubation at 31°C for 1-2 hr with pure PC liposomes (A. cholesterol depletion) or 0.52 mM cholesterol-PC liposomes (B. cholesterol enrichment). A. Cholesterol depletion 120- I 100- g 80“ 0I 60- 0 - I------1------1— I I m i p - - I 1 I I I I I l | ------1 I I I I I M | ------1------1-I I TT IT| 10-7 10-6 10-5 10-4 10-3 GABA concentration (M) B. Cholesterol enrichment 120i 100- < 80- O Control (n=9) A Enrichment (122%) (n=9) ▼ Enrichment (130%) (n=l2) r 10'^ 10-6 10-5 10-4 10 GABA concentration (M) The Influence of Membrane Cholesterol GABAon a Currents page 68 Chapter 4: Effect of Membrane Cholesterol on the Sensitivity of GABA ^ Receptors to GABA Figure 4.3 The effect of membrane cholesterol on the EC 50 values for GABA concentration-response relationship. Membrane cholesterol was manipulated by incubation at 31°C for 1-2 hr with pure PC liposomes (depletion) or cholesterol-PC liposomes (enrichment) at a final concentration of 0.52 mM cholesterol. The cholesterol content of control neurones was 0.299±0.012 pmoles/mg protein. Depletion Enrichment 15- I 10- < ( 12) 5- O 0-1 t 1 60 80 100 120 140 Membrane cholesterol content (% of control) * different from control neurones (f*<0.0001, Student's unpaired r-test) Numbers of neurones are shown in paretheses. The Influence of Membrane Cholesterol on GABA^ Currents page 69 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA 2) Membrane cholesterol alterations by methyl-P-cyclodextrin or its cholesterol inclusion complex (see section 3.2.3, Chapter 3) The currents induced by increasing the concentration of GABA on control, cholesterol-depleted and enriched neurones, by incubation with methyl-P- cyclodextiin or its cholesterol inclusion complex, are shown in figure 4.4. Cholesterol-depletion and enrichment resulted in a rightward shift of the GABA log.concentration-response curves from control neurones with no change in Hill slope (Figure 4.5). The same as incubation with liposomes, both cholesterol depletion and enrichment were associated with significant (P<0.0001) increases in the E C 5 0 values for the GABA log.concentration- response relationship (Figure 4.6). In control neurones, the GABA E C 5 0 value was 5.15+0.14 pM (n=9). In depleted neurones (53.9±2.53% of control by incubation with 10 mM methyl-P-cyclodextrin for 30 min), the GABA E C 5 0 value was 19.67+0.74 pM (n=9). In enriched neurones (235±5.40% of control by incubation with 0.3 mM cholesterol-methyl-P- cyclodextrin complex for 30 min), the GABA E C 5 0 value was 18.62+1.29 pM (n=8). The Influence of Membrane Cholesterol onGABA a Currents page 70 ______Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivity ofa GABA Receptors to GABA Figure 4.4 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on GABA a currents in acutely dissociated hippocampal neurones. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl- P-cyclodextrin (cholesterol depletion) or 0.15 mM cholesterol-methyl-p-cyclodextrin complex (enrichment) for 20 min. (A) Control neurone, (B) depleted neurone (56% of control), (C) enriched neurone (182% of control). G A B A (p M ) 0.1 0.3 1 3 10 30 100 300 1()00 IT < f L 20 s A. Control neurone G A BA (p M ) 0.1 0.3 1 3 10 30 100 300 1000 T 1 c 20 s B. Depleted neurone GABA (pM) 0.1 0.3 1 3 10 30 100 300 1000 FL ' 20 s C. Enriched neurone The Influence of Membrane Cholesterol on GABA^ Currents page 71 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA ofa Receptors to GABA Figure 4.5 The effect of membrane cholesterol alterations by methyl-(3- cyclodextrin on GABA concentration-response relationship. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (A. cholesterol depletion) or 0.15 mM cholesterol-methyl-p-cyclodextrin complex (B. cholesterol enrichment) for 5-30 min. A. Cholesterol depletion 120- c a 100- i 8 0 - Ü 6 0 - Control (n=9) .g 4 0 - Depletion (72%) (n=7) C3X Depletion (67%) (n=7) 20 - O Depletion (56%) (n=6) 0 - n Depletion (54%) (n=9) I------1—I I I 11 ii|------1—I -r rri ii|------1—i i i 11 ii|------1—i i i 11 ii| 0-7 10-6 10-5 10" * 10-3 GABA concentration (M) B. Cholesterol enrichment 120-1 c a 100- g < a 6 0 - O Control (n=9) .g A Enrichment (136%) (n=8) X 4 0 - I ▼ Enrichment (157%) (n=9) 20 - ♦ Enrichment (182%) (n=9) 0 ■ Enrichment (235%) (n-8) r 1—1 111111- 1— I I 11111] 10-6 10-5 10-4 10- 10-= GABA concentration (M) The Influence of Membrane Cholesterol GABAaon Currents page 72 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA Figure 4.6 The effect of membrane cholesterol on the EC 50 values for GABA concentration-response relationship. Membrane cholesterol was manipulated by incubation at 3TC with 10 mM methyl-p-cyclodextrin (depletion) for 5-30 min or cholesterol-methyl-P-cyclodextrin complex (enrichment) at a final concentration of 0.15 mM cholesterol for 5-20 min and 0.30 mM cholesterol for 30 min. The cholesterol content of control neurones was 0.319±0.009 pmoles/mg protein. Depletion Enrichment 25 n 20 - I 15- < 10- O 5 0 I T T T T T 50 100 150 200 250 Membrane cholesterol content (% of control) * different from control neurones (P<0.0001, Student's unpaired Mest) Numbers of neurones are shown in paretheses. The Influence of Membrane Cholesterol onGABAa Currents page 73 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA 4.2.2 Restoration of membrane cholesterol and GABA EC 50 value following depletion After cholesterol depletion by incubation with 10 mM methyl-P-cyclodextrin for 20 min, membrane cholesterol and also GABA EC 50 could be restored by incubation with 0.1 mM cholesterol-methyl-P-cyclodextrin complex for 1-10 min (Figure 4.7). 4.2.3 Effect of membrane cholesterol on the current-voltage relationship GABA whole-cell current-voltage relationships were constructed from the peak amplitudes of responses evoked by 3 and 10 pM GABA in control, depleted (56%) and enriched (182%) neurones (Figure 4.8) after incubation with methyl-P- cyclodextrin or its cholesterol inclusion complex, respectively, for 20 min. Although cholesterol depletion and enrichment reduced the amplitudes of GABA a currents in all neurones examined, there was no dramatic effect on the shape of GABA current- voltage relationship over the voltage range -30 to +30 mV (n=6-9). The GABA reversal potentials calculated from evoked current induced by 3 and 10 pM GABA in control, depleted and enriched neurones were not significantly different (P=0.75 and 0.57, respectively. One-way ANOVA, n=6-9). Evoked currents induced by 3 pM GABA in control, depleted and enriched neurones reversed at 0.89±0.30, 1.18±0.36 and 0.73±0.53 mV, respectively. Evoked currents induced by 10 pM GABA in control, depleted and enriched neurones reversed at 1.18±0.35, 1.20±0.43 and 0.69±0.39 mV, respectively. The Influence of Membrane Cholesterol onGABA a Currents page 74 Chapter 4: Effect of Membrane Cholesterol on the Sensitivity ofGABA^ Receptors to GABA Figure 4.7 Restoration of membrane cholesterol and GABA EC 50 values following cholesterol depletion. Membrane cholesterol was depleted by incubation at 31°C for 20 min with 10 mM methyl-p-cyclodextrin and subsequently restored by incubation at 31°C for 1-10 min with cholesterol-methyl-p-cyclodextrin complex at a final concentration of 0.1 mM cholesterol. 200 ■30 -20-20 Q o 150- -10 o C _co o ^ 100 O NS NS E (U 50- - Cholesterol content (n=3-6) Cholesterol depletion repletion 0 10 20 30 Incubation time (min) nonsignificance, * P<0.05, ** P<0.0 0 5 ,+**P<0.0001 , from control neurones, (Student's unpaired r-test) The Influence of Membrane Cholesterol on GABA^ Currents page 75 Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivityo/G A B A aReceptors to GABA Figure 4.8 The effect of membrane cholesterol on the GABA a receptor current- voltage relationship. Membrane cholesterol was manipulated by incubation at 31°C for 20 min with 10 mM methyl-p-cyclodextrin (depletion, 56% of control) or 0.15 mM cholesterol-methyl-P-cyclodextrin (enrichment, 182% of control). The neurones were voltage clamped between -30 to 4-30 mV. (A) 3 pM GABA, (B) 10 pM GABA. A. 3 pM GABA GABA-induccd current Current-voltage relationship "É I Control neurone 1500-, - I— " 2 0 s _ _ JV J c_ 1000 - 500- Holding potential (mV) Depleted neurone 20 30 JV_ JL -500- Control (n=9) E n rich ed n eu ron e - 1000- Depleted (n=6) _ j_JU -1500-1 Enriched (n=7) GABAa current (pA) -30 -20 -10 0 +10 +20 +30 Holding Potential (mV) B. 10 pM GABA GABA-induced current Control neurone Current-voltage relationship L 20 s iJ 2500-, 2000- 1500- 1000- Depleted neurone 500- Holding potential (mV) 1 1 JT -y— -30 20 3( -1000- Control (n=6) -1500- Depleted (n=6) Enriched neurone -2000- _ XJ -2500- Enriched (n=7) GABAa current (pA) -30 -20 -10 0 +10 +20 +30 Holding Potential (inV) The Influence of Membrane Cholesterol onGABAa Currents page 76 Chapter 4: Effect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA 4.2.4 Effect of acute application of cholesterol on GABAa currents The maximum concentration of cholesterol that could be achieved in 0.1% DMSO in PSS solution was 4 pM. Acute application of I and 4 pM cholesterol, in the same U- tube solution as the applied GABA, had no effect on the GABAa currents. The EC 50 values for the GABA log.concentration-response relationship remained unchanged at 5.04+0.21 pM (n=9) in the absence of cholesterol compared with 4.92±0.21 pM (n=9) and 5.52±0.24 pM (n=9) in the presence of 1 and 4 pM cholesterol, respectively (Figure 4.9). The Influence of Membrane Cholesterol onGABA a Currents page 77 Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivity of GABA^ Receptors to GABA Figure 4.9 The effect of acute application of cholesterol on the GABA concentration-response curves. Acute co-application of 1 and 4 p,M cholesterol with GABA did not affect GABA a currents in acutely dissociated hippocampal neurones. 120-, c 100- D. % 80- 60- O GABA alone (n=9) 40- A GABA + 1 laM cholesterol (n-9) 20 - ■ GABA + 4 cholesterol (n=9) I I t I M | T \ I I I I M| 10 10-6 10-5 10-4 10': GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 78 Chapter 4: Ejfect of Membrane Cholesterol on the SensitivityGABA of a Receptors to GABA 4.3 Discussion In the present study, the modulatory role of membrane cholesterol on the sensitivity of GABAa receptors to GABA was investigated in acutely dissociated rat hippocampal neurones. The two favoured methods, incubation with liposomes prepared from asolectin or pure PC with or without cholesterol and incubation with methyl-P- cyclodextrin or its cholesterol inclusion complex, were employed to manipulate membrane cholesterol of these neurones. Greater alterations of membrane cholesterol, down to 54% of control (depletion) or up to 235% of control (enrichment), were obtained by incubation with methyl-P-cyclodextrin or its cholesterol inclusion complex, respectively, than by incubation with pure PC liposomes or cholesterol-PC liposomes, down to 78% of control (depletion) or up to 130% of control (enrichment), respectively (see Chapter 3 for discussion). Unexpectedly, both depletion and enrichment of membrane cholesterol by these two methods resulted in significant (P<0.0001) increases of GABA EC 50 values (Figure 4.3 and 4.6). Membrane cholesterol depletion or enrichment altered neither the shape nor the reversal potential of whole-cell GABA current-voltage relationship (Figure 4.8), indicating that the effect of membrane cholesterol on GABA sensitivity of GABAa currents was not voltage dependent. To determine whether this decrease in the sensitivity of GABAa receptors to GABA was due to the membrane cholesterol alterations or to the effects of methyl-p- cyclodextrin, cholesterol depletion followed by repletion was explored. After depletion by incubation with 10 mM methyl-p-cyclodextrin for 20 min, membrane cholesterol as well as GABA EC 50 values could be restored by incubation with 0.1 mM cholesterol-methyl-P-cyclodextrin for 1-10 min (Figure 4.7). Since, the neurones were exposed to methyl-P-cyclodextiin throughout the depletion (10 mM) and repletion (1 The Influence of Membrane Cholesterol onGABA a Currents page 79 ______Chapter 4: Ejfect of Membrane Cholesterol on the Sensitivity of aGABA Receptors to GABA mM) procedures, there was clearly no cholesterol-independent effect of the methyl-13- cyclodextrin itself. The functions of many membrane enzymes and receptors are modulated by cholesterol (Yeagle, 1991; Gimpl et a l, 1997). Some of these membrane proteins, Na"^, K^-ATPase (Yeagle et a l, 1988), Na'^-Ca^'^ exchanger (Vemuri & Phillipson, 1989) and the GABA transporter (Shouffani & Kanner, 1990), have been reported to show decreased activities following either an increase (enrichment) or decrease (depletion) of the membrane cholesterol from the native cholesterol levels. In the absence of cholesterol these membrane proteins had little or no activity (Yeagle, 1991). The same effects of membrane cholesterol were found on the GABAa receptor of acutely dissociated rat hippocampal neurones in the present study. Cholesterol likely modulates the functions of these membrane proteins by more than one mechanism; for example, cholesterol may alter the bulk biophysical properties of the membrane, such as fluidity or it may modulate membrane proteins via specific cholesterol-protein interactions (Yeagle, 1993). These mechanisms may underline for the effect of cholesterol on the GABA sensitivity of GABAa receptors. However, further investigations need to be carried out to prove this hypothesis. The sites of action of cholesterol on GABAa receptors are likely to be in the plasma membrane since the acute co-application of GABA with cholesterol did not affect the GABA EC50 values (Figure 4.9). The Influence of Membrane Cholesterol on GABA^ Currents page 80 CHAPTER 5 EFFECT OF MEMBRANE CHOLESTEROL ON THE POTENTIATION OF GABAa c u r r e n t s BY NEUROACTIVE STEROIDS The Influence of Membrane Cholesterol on GABA^ Currents page 81 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids 5.1 Introduction Many steroids can exert allosteric modulation of the GABAa receptors (Lambert et al., 1995). Since these steroids have their structures related to cholesterol, there is a possibility of an interaction of the cholesterol in the plasma membrane with these steroids at the GABAa receptor. The first report of such an interaction by Bennett & Simmonds (1996) related to the enhancement of -flunitrazepam binding by pregnanolone. Whether or not analogous interactions of cholesterol and neuroactive steroids could be seen on electrophysiological responses to GABA is investigated in this chapter. The progesterone metabolites pregnanolone (5P-pregnan-3a-ol-20-one) and allopregnanolone (5a-pregnan-3a-ol-20-one) and the synthetic analogue alphaxalone (5a-pregnan-3a-ol-ll, 20-dione), which are very effective potentiators of GABA at the GABAa receptor (Simmonds, 1991; Lambert et al., 1995), were selected for this study. It was demonstrated in Chapter 4 that both membrane cholesterol depletion and enrichment decreased the sensitivity of the GABAa receptor to GABA, i.e. increased the GABA EC50 of the GABA concentration-response curve. To determine any effects of cholesterol on neuroactive steroid potency independent of an underlining change in GABA sensitivity, a ratio of E C 5 0 values for GABA in the presence and absence of neuroactive steroids was determined from each experiment. The concentrations of neuroactive steroids were chosen to cause a significant but submaximal shift of the GABA concentration-response curve to the left in control neurones. These concentrations were 0.3 and 1 |xM pregnanolone, 1 pM allopregnanolone and 1 pM alphaxalone. were Experiments[also performed to find out whether any effects of cholesterol on the potentiation of GABA by neuroactive steroids could also be seen with brief The Influence of Membrane Cholesterol onGABA a Currents page 82 Chapter 5: Effect on the PotentiationGABCurrents of by Neuroactive Steroids elevations of cholesterol in the medium, under conditions where little accumulation in the membranes would be expected. The Influence of Membrane Cholesterol onGABA a Currents page 83 Chapter 5: Ejfect on the PotentiationGABCurrents of by Neuroactive Steroids 5.2 Experimental protocols and results 5.2.1 Effect of membrane cholesterol on the potentiation of GABAa currents hy neuroactive steroids 1) Membrane cholesterol alterations by incubation with asolectin liposomes with or without cholesterol (see section 3.2.2, Chapter 3) Typical traces of GABA a currents from control, cholesterol-depleted (82% of control) and enriched (127% of control) neurones (Figure 5.1) show enhancements of the GABA a currents by 1 pM pregnanolone that are the clearly smaller in the enriched neurone but did not change ini depleted neurone. The GABA log.concentration-response relationships in the absence and presence of 1 pM pregnanolone of all the neurones are shown in figure 5.2. An analogous set of data for 1 pM alphaxalone is shown in figure 5.3. Membrane cholesterol enrichment also decreased the potentiations of GABA a currents by 0.3 pM pregnanolone and 1 pM allopregnanolone (Figure 5.4). The Influence of Membrane Cholesterol on GABA^ Currents page 84 ______Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.1 The effect of membrane cholesterol alterations by liposomes on the potentiation of GABAa currents by 1 pM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C with asolectin liposomes without cholesterol (depletion) or 0.52 mM cholesterol-asolectin liposomes (enrichment) for 1 hr. (A) Control neurone, (B) depleted neurone (82% of control), (C) enriched neurone (127% of control). A. Control neurone GABA (^iM) 0.1 0.3 1 3 10 30 100 300 1000 + Picgnaiiolonc “ - ■"IT' ' - - - -r - - (1 hM) f r r L 20 s B. Depleted neurone G A B A (jiM ) OU _ 0.3 1 3 10 30 100 300 1000 + P regnanolone - r ' r r r ' (1 HM) \ f r r r r I' 20 s C. Enriched neurone G A BA (n M ) OU 0.3 1 3 10 30 100 300 1000 + P regnanolone ------! — ." (1 nM) ■ T " r r L 20 s Note: All the control GABA responses were obtained first and those in the presence of drug subsequently. The Influence of Membrane Cholesterol on GABA^ Currents page 85 Chapter 5: Effect on the Potentiation of GABA^ Currents by Neuroactive Steroids Figure 5.2 The effect of membrane cholesterol alterations by liposomes on the concentration-response relationship for the potentiation of GABAa currents by 1 pM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C for 1 hr with asolectin liposomes (A. cholesterol depletion) or 0.52 mM cholesterol- asolectin liposomes (B. cholesterol enrichment). The potentiation of GABAa currents by 1 pM pregnanolone was not affected by cholesterol depletion (82% of control) but was reduced by cholesterol enrichment (127% of control). The effects of cholesterol depletion and enrichment on the GABA alone curves have been described in section 4.2.1, Chapter 4. A. Cholesterol depletion 120-1 100- 80- 60- 40- O Control, GABA alone (n=9) V Control, GABA+Pregnanolone 20- • Depleted, GABA alone (n=l 1 ) 0- ▼ Depleted, GABA+Pregnanolone I------1—I- I i-n ii'i----- 1—I I T I I ii|------1—I I I I Mi| I—I I I 11 ii| 10-7 10-6 1Q-5 10-4 10-3 GABA concentration (M) B. Cholesterol enrichment 120n a 100- 80- 60- Control, GABA alone (n=9) X 40- Control, GABA+Pregnanolone I 20- Enriched, GABA alone (n-9) 0- Enriched, GABA+Pregnanolone I------1— ...... 1 I I I ! Il| 1...... 1 I » I I M l) 10'^ 10® 10 ® 10 " 10 ® GABA Concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 86 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.3 The effect of membrane cholesterol alterations by liposomes on the concentration-response relationship for the potentiation of GABAa currents by 1 pM alphaxalone. Membrane cholesterol was manipulated by incubation at 31°C for 1 hr with asolectin liposomes (A. cholesterol depletion) or 0.52 mM cholesterol-asolectin liposomes (B. cholesterol enrichment). The potentiation of GABAa currents by 1 pM alphaxalone was not affect by cholesterol depletion (82%) but was reduced by cholesterol enrichment (127%). A. Cholesterol depletion 120- 100- 8 0 - 6 0 - 4 0 - Control, GABA alone (n=9) Control, GABA+Alphaxalone 20- Depleted, GABA alone (n=7) 0- Depleted, GABA+Alphaxalone I------1 t I i M ll| I I I I n i| 1 ■! r r n n] ■ i i i m iii| 10'^ 10'® 10® lO'* 10® GABA concentration (M) B. Cholesterol enrichment 120-1 100- 8 0 - a 6 0 - O Control, GABA alone (n=9) 4 0 - □ Control, GABA+Alphaxalone 20- • Enriched, GABA alone (n=9) 0- ■ Enriched, GABA+Alphaxalone I 1— I T t I I iT| I— r - r 10-" 10® 10® 10’^ 10 -3 GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 87 ______Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.4 The effect of membrane cholesterol alterations by liposomes on the concentration-response relationship for the potentiation of GABAa currents by 0.3 pM pregnanolone and 1 pM allopregnanolone. Membrane cholesterol was manipulated by incubation at 31°C for 1 hr with 0.52 mM cholesterol-asolectin liposomes. The potentiation of GABAa currents by 0.3 pM pregnanolone (A) and 1 pM allopregnanolone (B) was reduced by cholesterol enrichment (127%). A. 0.3 pM Pregnanolone 120- 100- 8 0 - 6 0 - O Control, GABA alone (n=6) 4 0 - % O Control, GABA+Pregnanolone 20- • Enriched, GABA alone (n=7) 0- ♦ Enriched, GABA+Pregnanolone rTTTT------T T q ------1— I I I I I m -3 10'^ 10® 1 0 ® 10 " 10 GABA concentration (M) B. 1 pM Allopregnanolone 120n 100 - 80- Ü 6 0 - 4 0 - Control, GABA alone (n=12) 20- Control, GABA+Allopregnanolone Enriched, GABA alone (n=12) 0- Enriched, GABA+Allopregnanolone I 1 1 I I I I M | 1------1 I I I M l | 1 1 I I I I l l | 1 1 I I I I l l | 10-/ 10 ® 10 ® 10'^ 10 ® GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 88 Chapter 5: Ejfect on the PotentiationGABA o f a Currents by Neuroactive Steroids For statistical analysis of the effects of membrane cholesterol on the potencies of the neuroactive steroids as GABA potentiators, a ratio of EC 50 values for GABA in the presence and absence of neuroactive steroid was determined from each experiment. Table 5.1 shows that the mean±SEM of the EC50 ratios for each of the neuroactive steroids is significantly less than 1 (P<0.01, Student’s r-test), as expected for a GABA potentiator, and that the difference from unity is significantly smaller in cholesterol-enriched neurones than in control neurones for each neuroactive steroid. Thus, the potency of each of the neuroactive steroids was reduced by cholesterol enrichment. However, with the rather small decrease in membrane cholesterol (to 82% of control) in depleted neurones, the GABA EC 50 ratios for 1 \xM pregnanolone and 1 p,M alphaxalone were not different from the corresponding values in control neurones (Table 5.1). Table 5.1 The effect of membrane cholesterol alterations by asolectin liposomes on GABA EC 50 ratios for the potentiation of GABAa currents by neuroactive steroids. Membrane cholesterol was manipulated by incubation at 31°C for 1 hr with asolectin liposomes or 0.52 mM cholesterol-asolectin-liposomes. Numbers of experiments are shown in parentheses. GABA EC50 ratio ((GABA+neuroactive steroid)/GABA) Neuroactive steroid Control Depleted Enriched (82% of control) (127% of control) 0.3 pM Pregnanolone 0.225±0.043 (6 )- 0.483±0.075* (7) 1 pM Pregnanolone 0.175±0.011 (9) 0.164±0.014'^^(11) 0.370±0.058** (9) 1 pM Allopregnanolone 0.097±0.012(12) - 0.248±0.045** (12) 1 pM Alphaxalone 0.279±0.021 (9) 0.280±0.025^^ (7) 0.441±0.062* (9) NS nonsignificantly different from control, * f<0.05, ** P<0.01, Student’s r-test. The Influence of Membrane Cholesterol onGABA a Currents page 89 Chapter 5: Ejfect on the Potentiationo/GABA a Currents by Neuroactive Steroids 2) Membrane cholesterol alterations by incubation with pure PC liposomes with or without cholesterol (see section 3.2.2, Chapter 3) The experiments with 1 pM pregnanolone were repeated on neurones in which asolectin was replaced by pure PC in the liposomal cholesterol environment procedure. After incubation with 0.52 mM cholesterol-pure PC liposomes for 1 and 2 hr, a reduction of the potentiation of GABAa currents by 1 pM pregnanolone by cholesterol enrichment (122% and 130% of control, respectively) was again found (Table 5.2). Also the slight decrease in membrane cholesterol (84% and 78% of control) by incubation with pure PC liposomes for 1 and 2 hr again resulted in the GABA ECso ratios for 1 pM pregnanolone being not different from the corresponding values in control neurones (Table 5.2). Table 5.2 The effect of membrane cholesterol alterations by pure PC liposomes on GABA EC 50 ratios for the potentiation of GABAa currents by 1 pM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C for 1 and 2 hr with pure PC liposomes or 0.52 mM cholesterol-pure PC liposomes. Numbers of experiments are shown in parentheses. Membrane cholesterol content GABA EC50 ratio (% of control) ((GABA + 1 )llM pregnanolone)/GABA) 78% 0.157±0.015^^(9) 84% 0.155±0.016^^ (9) Control (100%) 0.175±0.011 (9) 1 2 2 % 0.287±0.033* (9) 130% 0.316±0.033*(12) nonsignificantly different from control, * P<0.001, Student’s r-test. The Influence of Membrane Cholesterol onGABA a Currents page 90 Chapter 5: Effect on the PotentiationGABCurrents o f by Neuroactive Steroids 3) Membrane cholesterol alterations by incubation with methyl-13- cyclodextrin or its cholesterol inclusion complex (see section 3.2.3, Chapter 3) The potentiations of GABAa currents by 1 pM pregnanolone in control, depleted (56% of control) and enriched (235% of control) neurones are shown in figure 5.5. The potentiation of GABAa currents by 1 pM pregnanolone resulted in a leftward shift of the GABA log.concentration- response curve (Figure 5.6). The leftward shift of the log.concentration- response curve in depleted neurones was higher than in control neurones. In contrast to the cholesterol depletion, the shift was smaller in enriched neurones than in control neurones. The same results were also found for the potentiation of GABAa currents by 1 pM alphaxalone (Figure 5.7). The GABA EC50 ratios for 1 pM pregnanolone of 0.197±0.014 (n=9) in control neurones and 0.116+0.014 (n=9) at the lowest membrane cholesterol (54% of control) represented the greatest enhancement of potentiation (P<0.001) (Figure 5.8). At a lesser depletion of cholesterol (72% of control) similar to that obtained by incubation with liposomes, the GABA E C 5 0 ratio for 1 pM pregnanolone of 0.164±0.012 was, as in the liposome experiments, not significantly different from control neurones. In contrast to the cholesterol depletion, the ratio of 0.303±0.029 (n= 8) obtained at the highest cholesterol (235% of control) represented a significant decline (P< 0 .0 1 ) in the potentiation of GABAa currents by 1 pM pregnanolone (Figure 5.8). Membrane cholesterol depletion and enrichment also increased and decreased, respectively, the potentiation of GAB A A currents by 1 pM alphaxalone (Table 5.3). All the E C 5 0 ratios for pregnanolone and alphaxalone are summarised in table 5.3. The Influence of Membrane Cholesterol onGABA a Currents page 91 Chapter 5: Effect on the PotentiationGABCurrents of by Neuroactive Steroids Figure 5.5 The effect of membrane cholesterol alterations by methyl-^- cyclodextrin on the potentiation of GABA a currents by 1 pM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl- p-cyclodextrin (cholesterol depletion) for 20 min or 0.30 mM cholesterol-methyl-P- cyclodextrin complex (enrichment) for 30 min. (A) Control neurone, (B) Depleted neurone (56% of control), (C) Enriched neurone (235% of control). A. Control neurone G A B A (^ M ) 0.1 _ 0.3 1 3 10 30 100 300 1000 + Pregnanolone 2- (1 nM) y i r r L 20 s B. Depleted neurone G A B A (n M ) 0.1 0.3 1 3 10 30 100 300 1000 + P r e g n a n o lo n e “ ^ __ (1 nM) r T 1 _ 20 s 20 s C. Enriched neurone GABA(nM) OU 0.3 1 3 10 30 100 300 1000 + Pregnanolone " - (1 ^M) V JT r T 20 s 20 s Note: All the control GABA responses were obtained first and those in the presence of drug subsequently. The Influence of Membrane Cholesterol on GABA^ Currents page 92 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.6 The effect of membrane cholesterol alterations by methyl-P- cyclodextrin on the concentration-response relationship for the potentiation of GABAa currents by 1 piM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (depletion) for 20 min or 0.30 mM cholesterol-methyl-p-cyclodextrin complex (enrichment) for 30 min. The potentiation of GABAa currents by 1 pM pregnanolone was enhanced by (A) cholesterol depletion (56% of control) but was reduced by (B) cholesterol enrichment (235% of control). The effects of cholesterol depletion and enrichment on the GABA alone curves have been described in section 4.2.1, Chapter 4. A. Cholesterol depletion 120- 100- < 8 0 - o 60 E O Control, GABA alone (n=9) X 40 A Control, GABA-rPregnanolone S 20 • Depleted, GABA alone (n=6) 0 A Depleted, GABA+Pregnanolone I 1 T I I 1 M l) 1----- 1 I I I T l l [ 1------1 I M I l l | 1 1 I I I I M | 10-7 10-6 10-6 10-^ 10-6 GABA concentration (M) B. Cholesterol enrichment 120i c §. 100- < 8 0 - Ü 6 0 - I 4 0 - O Control, GABA alone (n=9) □ Control, GABA+Pregnanolone ^ 20- • Enriched, GABA alone (n=8) 0- ■ Enriched, GABA+Pregnanolone I------1-----1 I I I I I l | ------I----- 1 T I I I IT|------1----- 1 I I I I l l | ------1----- 1 I I I I l l | 10-7 10-6 10-5 10-4 10-3 GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 93 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.7 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on the concentration-response relationship for the potentiation of GABAa currents by 1 pM alphaxalone. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-g-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl-p-cyclodextrin complex (enrichment) for 20 min. The potentiation of GABAa currents by 1 pM alphaxalone was enhanced by (A) cholesterol depletion (56% of control) but was reduced by (B) cholesterol enrichment (182% of control). A. Cholesterol depletion 120- 100- 80- cd e 6 0 - X c3 / / O Control, GABA alone (n=9) jg V Control, GABA+Alphaxalone Depleted, GABA alone (n-7) ^ ▼ Depleted, GABA+Alphaxalone I 1 1 I I M ll| 1 1 I I I lll| 1 1 I I I ril| 1 till TTT| 10-7 10-6 10-5 1Q-4 1Q-3 GABA concentration (M) B. Cholesterol enrichment 120-1 100 - c a 80- 60- O Control, GABA alone (n=9) 40- V Control, GABA+Alphaxalone 20- • Enriched, GABA alone (n=7) 0 ▼ Enriched, GABA+Alphaxalone r I 111 ii| 1 10- 1 0 - 6 1 0 5 1 0 -4 10 GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 94 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.8 The effect of membrane cholesterol alterations by methyl-(3- cyclodextrin on the GABA EC 50 ratios for the potentiation of GABAa currents by IpM pregnanolone. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (depletion) or 0.15-0.30 mM cholesterol-methyl- P-cyclodextrin complex (enrichment) for 5-30 min. Each point represents the mean±S.E.M. from 6-9 neurones. The cholesterol content of control neurones was 0.319±0.009 pmoles/mg protein (n=12). Depletion Enrichm ent 0.35-1 ÜI 0.30- c 0O 0.25- § c 0 .20 - D- 0.15- o1 O 0. 10- 0.05- * P<0.05, ** P<0.01, *** P<0.001 0 .00 ^ (from control. Student's t-test) 50 75 100 125 150 175 200 225 250 Membrane cholesterol content (% of control) The Influence of Membrane Cholesterol onGABA a Currents page 95 Chapter 5: Ejfect on the Potentiation o f Ga ABA. Currents by Neuroactive Steroids Table 5.3 The effect of membrane cholesterol alterations by methyl-P- cyclodextrin on GABA EC 50 ratios for the potentiation of GABAa currents by neuroactive steroids. Membrane cholesterol was manipulated by incubation at 31°C for 5-30 min with 10 mM methyl-p-cyclodextrin (depletion) or 0.15-0.3 mM cholesterol-methyl-p-cyclodextrin complex (enrichment). Numbers of experiments are shown in parentheses. Membrane cholesterol GABA EC50 ratio ((GABA+neuroactive steroid)/GABA) content (% of control) 1 pM Pregnanolone 1 pM Alphaxalone 54% 0.116±0.014*** (9) - 56% 0.115±0.011*** (6) 0.185±0.008** (7) 67% 0.134±0.014**(7) - 72% 0.164±0.012^^ (7) - Control (100%) 0.197±0.014 (9) 0.275±0.015 (9) 136% 0.250±0.021* (8) - 157% 0.283±0.029* (9) - 182% 0.293±0.024** (9) 0.358±0.022** (7) 235% 0.303±0.029** (8) - The Influence o f Membrane Cholesterol onGABA a Currents page 96 Chapter 5: Ejfect on the PotentiationGABA of a Currents by Neuroactive Steroids 5.2.2 Restoration of membrane cholesterol and GABA EC 50 ratios following depletion After cholesterol depletion to 56% of control incubation with 0.1 mM cholesterol- methyl-P-cyclodextrin complex for 1-10 min restored the cholesterol to 103% of control (see section 3.2,4, Chapter 3) and increased the GABA EC 50 ratio for 1 pM pregnanolone from 0.115±0.011 (n=6) to 0.191±0.020 (P=0.80, n=6, compared with control value of 0.197±0.014 (n=9)) (Figure 5.9). 5.2.3 Effect of acute application of cholesterol on the potentiation of GABAa currents hy pregnanolone. Acute application of 1 pM cholesterol for about 5 sec, in the same U-tube solution as the applied GABA, had no effect on the GABAa currents, the EC 50 values for GABA remaining unchanged at 4.92+0.21 pM (P=0.69, n=9) in the presence of 1 pM cholesterol compared with 5.04+0.21 pM (n=9) in the absence of cholesterol. Additionally, the acute application of cholesterol did not significantly affect the potentiation of GABAa currents by 1 pM pregnanolone (Figure 5.10). The Influence of Membrane Cholesterol onGABA a Currents page 97 Chapter 5: Ejfect on the Potentiation of GABA^ Currents by Neuroactive Steroids Figure 5.9 Restoration of membrane cholesterol and GABA EC 50 ratios for the potentiation of GABAa currents by 1 pM pregnanolone in cholesterol depleted neurones (56% of control). Membrane cholesterol was depleted by incubation at 31°C for 20 min with 10 mM methyl-P-cyclodextrin and subsequently restored by incubation at 31°C for 1-10 min with cholesterol-methyl-P-cyclodextrin complex at a final concentration of 0.1 mM cholesterol. Each point represents the meaniS.E.M. from 6-9 neurones. 150-1 Cholesterol depletion Repletion - EC 5 0 ratio - 0.20 ^ ^ 100 -0.10 3 -0.05 Cholesterol content 10 20 Incubation time (min) nonsignificance, * f<0.05, ** P<0.01, *** f<0.001, from control neurones, (Student's unpaired /-test) The Influence of Membrane Cholesterol on GABA^ Currents page 98 Chapter 5: Effect on the PotentiationGABA of a Currents by Neuroactive Steroids Figure 5.10 The effect of acute application of cholesterol on the concentration- response curves for the potentiation of GABA a currents by 1 p-M pregnanolone. Acute co-application of 1 pM cholesterol with GABA did not affect the potentiation of GABA a currents by 1 pM pregnanolone (n=5). 120-1 (U c 100- 00a < 8 0 - O 6 0 - 15 B 4 0 - 20- O GABA alone A GABA + 1 ^iM Pregnanolone 0 □ GABA + 1 |aM Cholesterol + 1 Pregnanolone r T 1 1- 1- IT T I T 1 I I I I I I T 1 I I 1 t I) 10 10-6 10-5 lO-'* 10-= GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 99 Chapter 5: Effect on the Potentiation of Ga ABA Currents by Neuroactive Steroids 5.3 Discussion The potentiation of GABAa currents by pregnanolone, allopregnanolone and alphaxalone was substantially reduced, as shown by the increase of G ABA EC 50 ratios for neuroactive steroids, in cholesterol-enriched (127% of control) neurones by incubation with asolectin liposomes containing cholesterol (Table 5.1). The reduction of G ABA potentiation by 1 p.M pregnanolone was also found in cholesterol-enriched neurones by incubation with pure PC liposomes containing cholesterol (122-130% of control) (Table 5.2) or cholesterol-methyl-P-cyclodextrin complex (136-235% of control) (Table 5.3). After comparing the degree of the reduction of GAB A potentiation by these three cholesterol carriers, cholesterol- asolectin liposomes, cholesterol-pure PC liposomes and cholesterol-methyl-P- cyclodextrin complex, at about the same membrane cholesterol contents (127%, 130% and 136% of control, respectively), the reductions of the potentiation were not significantly different from each other (P=0.17, ANOVA) (Table 5.1-5.3). Thus, irrespective of the cholesterol carrier used to alter membrane cholesterol, cholesterol depletion was found to enhance and cholesterol enrichment to reduce, the potentiation of GABAa currents by neuroactive steroids. The use of methyl-p- cyclodextrin as the cholesterol carrier clearly had no additional direct effect of its own, since the repletion of membrane cholesterol following depletion reduced the enhancement of the GAB A potentiation by pregnanolone back to the control level (Figure 5.9). These results are compatible with a competition between cholesterol and the neuroactive steroids for their site(s) of action on the GABA a receptor. Nevertheless, other mechanisms of the interaction between cholesterol and neuroactive steroids cannot be excluded. For example, cholesterol may have restricted the access of neuroactive steroids to their recognition sites in the membrane or it may have had a The Influence of Membrane Cholesterol onGABA a Currents page 100 Chapter 5: Effect on the PotentiationGABCurrents of by Neuroactive Steroids more global allosteric action that could affect other G ABA potentiators in addition to the neuroactive steroids. This possibihty will be investigated in the next chapter. If specific protein binding sites for cholesterol are involved, they appear to be located on the transmembrane regions of the GABA a receptor since only alterations of membrane cholesterol and not acute application of extracellular cholesterol were effective (Figure 5.10). The Influence of Membrane Cholesterol onGABA a Currents page 101 CHAPTER 6 EFFECT OF MEMBRANE CHOLESTEROL ON THE MODULATION OF GABAa c u r r e n t s BY OTHER DRUGS The Influence of Membrane Cholesterol on GABA^ Currents page 102 Chapter 6: Effect on the ModulationGABA o f a Currents by Other Drugs 6.1 Introduction Many membrane proteins and receptors have been reported to be modulated by cholesterol (Jones & McNamee, 1988; Yeagle, 1991; Gimpl et at., 1997). Both membrane cholesterol depletion and enrichment also reduced the sensitivity of GABAa receptor to GAB A in acutely dissociated hippocampal neurones (see Chapter 4 for details). Membrane cholesterol enrichment also decreased the potentiation of GABAa currents by neuroactive steroids (see Chapter 5 for details). In contrast, cholesterol depletion increased the potentiation of GABAa currents by neuroactive steroids (see Chapter 5 for details). Following on from the observations in Chapter 5 that cholesterol reduced the potentiation of GABAa currents by neuroactive steroids, it was obviously of interest to investigate whether the same effects of cholesterol would be found on the other GAB A potentiators. Three potentiators, propofol, pentobarbital sodium and flunitrazepam that have been shown to have separate sites of action from that for neuroactive steroids on GABAa receptor (Prince & Simmonds, 1992; Lambert et al., 1995; Sieghart, 1995), were selected for this study. Additionally, three GAB A antagonists, bicuculline (competitive antagonist), picrotoxinin (noncompetitive antagonist) and pregnenolone sulphate (negative modulator, neuroactive steroid) were also investigated. The Influence of Membrane Cholesterol onGABA a Currents page 103 Chapter 6: Effect on the Modulation o/GABA^ Currents by Other Drugs 6.2 Experimental protocols and results 6.2.1 Effect of membrane cholesterol on the potentiation of GABAa currents hy drugs 1) Propofol The potentiations of GABA a currents by 5 |xM propofol in control, depleted and enriched neurones were shown in figure 6.1. After incubation with 10 mM methyl-p-cyclodextrin at 31°C for 20 min (see section 3.2.3, Chapter 3), the potentiation of GABA a currents by 5 pM propofol was not affected by membrane cholesterol depletion (56% of control) (Figure 6.2). In contrast to cholesterol depletion, after incubation with 0.15 mM cholesterol-methyl-p-cyclodextrin for 20 min (see section 3.2.3, Chapter 3), the potentiation of GABAa currents by 5 pM propofol was enhanced (P<0.001, n= 8 ) at the high level of cholesterol enrichment (182% of control) (Figure 6.2). The GABA EC 50 ratios for 5 pM propofol of these neurones are shown in table 6.1. However, at the low level of cholesterol enrichment (136% of control), by incubation with 0.15 mM cholesterol- methyl-P-cyclodextrin for 5 min. The GABA E C 5 0 ratio of 0.214+0.014 (n=10) in enriched neurones was not significant different (P=0.49) from 0.228±0.014 (n=10) in control neurones. The Influence o f Membrane Cholesterol onGABA a Currents page 104 Chapter 6: Effect on the Modulation of Ga ABA Currents by Other Drugs Figure 6.1 The effect of membrane cholesterol alterations by methyl-f- cyclodextrin on the potentiation of GABA a cuiTcnts by 5 |iM propofol. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p- cyclodextrin (cholesterol depletion) for 20 min or 0.15 mM cholesterol-methyl-P- cyclodextrin complex (enrichment) for 20 min. (A) Control neurone, (B) Depleted neurone (56% of control), (C) Enriched neurone (182% of control). A. Control neurone GABA(mM) 0.1 0.3 3 10 30 100 300 1000 + Propofol " (5nM) r T r B. Depleted neurone GABA(nlM) 0.1 0.3 3 10 30 100 300 1000 + Propofol * : ____ (5pM) nr IT r r C. Enriched neurone GABA (pM) 0.1 0.3 3 10 30 100 300 1000 + Propofol (5pM) f— r “ /■— r r r y r~ r f L Î L " 20; 20 s N ote: All the control GABA responses were obtained first and those in the presence of drug subsequently. The Influence of Membrane Cholesterol onGABA a Currents page 105 Chapter 6: Ejfect on the Modulation of GABA^ Currents by Other Drugs Figure 6.2 The effect of membrane cholesterol alterations by methyl-g- cyclodextrin on the concentration-response relationship for the potentiation of GABAa currents by 5 pM propofol. Membrane cholesterol was manipulated by incubation at 31°C for 20 min with 10 mM methyl-p-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl-P-cyclodextrin complex (enrichment). The potentiation of GABAa currents by 5 pM propofol was enhanced by (B) cholesterol enrichment (182% of control) but was not affected by (A) cholesterol depletion (56% of control). A. Cholesterol depletion 120-, c 8. 100- g 80- 0I 60- 15 .E O Control, GABA alone (n=10) X 40- 1 □ Control, GABA+Propofol 20 - • Depleted, GABA alone (n=9) 0 ■ Depleted, GABA+Propofol T ...... 1 10'^ 10'® 10® 10 10'= GABA concentration (M) B. Cholesterol enrichment 120n c a 100- g 80- oI 60- 15 O Control, GABA alone (n=IO) .EX 40- □ Control, GABA+Propofol 20 - • Enriched, GABA alone (n=9) 0 ■ Enriched, GABA+Propofol I I u m Î I I I M f l | 10 1 0 ® 1 0 ® 10 10- GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 106 Chapter 6: Effect on the Modulation o/GABA^ Currents by Other Drugs Incubation of neurones with liposomes (with or without cholesterol), which resulted in only small changes (82-127% of control) in membrane cholesterol (see section 3.2.2, Chapter 3), did not affect the potentiation of GABAa currents by 5 [iM propofol (Figure 6.3). The GABA EC 50 ratios for 5 propofol of 0.272±0.030 (n=10) in depleted neurones and of 0.329±0.062 (n=12) in enriched neurones were not significant different from 0.319±0.068 (n=12) in control neurones. Table 6.1 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on GABA EC 50 ratios for the modulation of GABAa currents by drugs. Membrane cholesterol was manipulated by incubation at 31°C for 20 min with 10 mM methyl-p-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl-p- cyclodextrin complex (enrichment). Numbers of experiments are shown in parentheses. GABA EC50 ratio ((GABA+modulator)/GABA) GABA modulator Control Depleted Enriched (56% of control) 182% of control) Potentiator 5 pM Propofol 0.228±0.014 (10) 0.19610.023'^^ (9) 0.12210.008* (9) 1 pM Flunitrazepam 0.516±0.022 (9) 0.49910.034^^ (9) 0.47610.028^^^ ( 8 ) 10 pM Flunitrazepam 0.47710.018(10) 0.39710.039^^^(10) 0.27610.017** ( 8 ) 5 pM Pentobarbital sodium 0.63910.022 (9) 0.61710.046^^^ ( 6 ) 0.68210.031'^^ ( 8 ) 20 pM Pentobarbital sodium 0.56310.023 ( 8 ) 0.51810.033^^ (8 ) 0.43710.013* (10) Antagonist 5 pM Bicuculline 7.5710.545 ( 8 ) 6.5410.671^^ (8) 4.5110.463* ( 8 ) 10 pM Picrotoxinin 9.1812.28 (9) 11.1812.61^^^ (7) 8.4411.14^^^(9) 20 pM Pregnenolone sulphate 2.1110.141 (10) 2.0010.082^^^ (1 0 ) 1.8710.172^^ ( 8 ) The Influence o f Membrane Cholesterol onGABA a Currents page 107 Chapter 6: Ejfect on the Modulation o/GABA^ Currents by Other Drugs Figure 6.3 The effect of membrane cholesterol alterations by liposomes on the concentration-response relationship for the potentiation of GABAa currents by 5 pM propofol. Membrane cholesterol was manipulated by incubation at 31°C for 1 hr with asolectin liposomes (A. cholesterol depletion) or 0.52 mM cholesterol-asolectin liposomes (B. cholesterol enrichment). The potentiation of GABAa currents by 5 pM propofol was not affected by (A) cholesterol depletion (82%) or by (B) cholesterol enrichment (127%). A. Cholesterol depletion 120-1 c I 100- 80- 0I 60- .g Control, GABA alone (n=12) X 4 0 - 1 Control, GABA+Propofol 2 0 - Depleted, GABA alone (n=10) 0 Depleted, GABA+Propofol 10'^ 10-6 -10-5 10-4 10 = GABA concentration (M) B. Cholesterol enrichment 120-1 I 100- Ë I 80- o 60- O Control, GABA alone (n=12) •i 40- cdX S □ Control, GABA+Propofol 2 0 - • Enriched, GABA alone (n=l 2) 0 ■ Enriched, GABA+Propofol T—I I n 111'l 10 lO'G 10 10 10 GABA concentration (M) The Influence of Membrane Cholesterol onGABA a Currents page 108 Chapter 6: Ejfect on the Modulation o f GABA Currents by Other Drugs 2) Pentobarbital sodium After incubation at 31°C for 20 min with 10 mM methyl-P-cyclodextrin or 0.15 mM cholesterol methyl-P-cyclodextrin complex (see section 3.2.3, Chapter 3), the potentiation of GABA a currents by 5 pM pentobarbital sodium was not affected by membrane cholesterol depletion or enrichment (Table 6.1). Although the potentiation of GABA a currents by 20 pM pentobarbital sodium was also not affected by cholesterol depletion, it was enhanced by cholesterol enrichment (Figure 6.4 and Table 6.1). The GABA ECso ratio for 20 pM pentobarbital sodium of 0.563±0.023 (n=8) in control neurones decreased to 0.437±0.013 (P<0.001, n=10) in enriched neurones (Table 6.1). 3) Flunitrazepam Membrane cholesterol in acutely dissociated hippocampal neurones was depleted (56% of control) or enriched (182% of control) by incubation at 31°C for 20 min with 10 mM methyl-P-cyclodextrin or 0.15 mM cholesterol methyl-p-cyclodextrin complex, respectively (see section 3.2.3, Chapter 3). Membrane cholesterol alterations did not affect the potentiation of GABA a currents by 1 pM flunitrazepam (Table 6.1). The potentiation of GABA a currents by 10 pM flunitrazepam was also not affected by cholesterol depletion but it was enhanced by cholesterol enrichment (Figure 6.5). The GABA E C 5 0 ratio for 10 pM flunitrazepam of 0.477±0.018 (n=10) in control neurones decreased to 0.276±0.017 (P<0.0001, n=8) in enriched neurones (Table 6.1). The Influence of Membrane Cholesterol on GABA^ Currents page 109 ______Chapter 6: Ejfect on the Modulation of GABA^ Currents by Other Drugs Figure 6.4 The effect of membrane cholesterol alterations by methyl-g- cyclodextrin on the concentration-response relationship for the potentiation of GABAa currents by 20 pM pentobarbital sodium. Membrane cholesterol was manipulated by incubation at 31°C for 20 min with 10 mM methyl-P-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl-P-cyclodextrin complex (enrichment). The potentiation of GABAa currents by 20 pM pentobarbital sodium was not affected by (A) cholesterol depletion (56% of control) but was enhanced by (B) cholesterol enrichment (182% of control). A. Cholesterol depletion 120i I 100- < 80- 60- E O Control, GABA alone (n=8) X 40- I O Control, GABA+Pentobarbital 20 - • Depleted, GABA alone (n=8) 0 ♦ Depleted, GABA+Pentobarbital T m !|- T 1 » i M ll| 10 10’® 10- 10 10-= GABA concentration (M) B. Cholesterol enrichment 120n c a 100- oI 60- O Control, GABA alone (n=8) E 40- : O Control, GABA+Pentobarbital s 20 - • Enriched, GABA alone (n=10) 0 ♦ Enriched, GABA+Pentobarbital 1 10- 10-G 10-" 10- 10’ = GAB A concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 110 Chapter 6: Ejfect on the Modulation of GABA^ Currents by Other Drugs Figure 6.5 The effect of membrane cholesterol alterations by methyl-g- cyclodextrin on the concentration-response relationship for the potentiation of GABA a currents by 10 p.M flunitrazepam. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl-P-cyclodextrin complex (enrichment) for 20 min. The potentiation of GABA a currents by 10 p,M flunitrazepam was not affected by (A) cholesterol depletion (56% of control) but was enhanced by (B) cholesterol enrichment (182% of control). A. Cholesterol depletion 0 120n I - 1004 Ü < 80- DQ 3 60- O Control, GABA alone (n=10) 40- X A Control, GABA+Flunitrazepam I 20 - • Depleted, GABA alone (n=l 0) 0- A Depleted, GABA+Flunitrazepam ■T"- r I T I T l l | 1 I I I I I l l | 1 I I I I I l l | I 1 I I I I t l | 10'^ 10- 10' 10-' 10-= GABA concentration (M) B. Cholesterol enrichment 120n I 100- < 80- CQ < 60- O Control, GABA alone (n=10) 40- V Control, GABA+Flunitrazepam 20- • Enriched, GABA alone (n=8) 0 ▼ Enriched, GABA+Flunitrazepam r 1-----1 I 1 I M l| 1---1 I M lll| T 1 I I I I l l | 1 10-^ 10-6 10-5 1Q-4 10-= GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 111 ______Chapter 6: Ejfect on the ModulationGABCurrents o f by Other Drugs 6.2.2 Effect of membrane cholesterol on the antagonism of GABAa currents hy the drugs 1) Bicuculline methochloride The antagonism of GABAa currents by 5 |xM bicuculline methochloride in control, depleted and enriched neurones is shown in figure 6 .6 . Bicuculline shifted the GABA concentration-response curve to the right without change in maximal GABA response (Figure 6.7). Although the antagonism of GABAa currents by bicuculline was not affected by cholesterol depletion, it was reduced by cholesterol enrichment (Figure 6.7). The GABA EC 50 ratios for 5 pM bicuculline were more than 1, as expected for GABA antagonist (Table 6.1). Cholesterol enrichment decreased the GABA E C 5 0 ratio for 5 pM bicuculline of 7.57±0.545 (n= 8 ) in control neurones to 4.51+0.463 in enriched neurones (P<0.001, n= 8) (Table 6.1). 2) Picrotoxinin Application of 10 pM picrotoxinin, in the same U-tube solution as the applied GABA, resulted in both rightward shift of GABA curve and a reduction in its maximum (Figure 6 .8 ). In contrast to bicuculline, both membrane cholesterol depletion and enrichment did not affect the inhibition of GABA a currents by 10 pM picrotoxinin. The GABA E C 5 0 ratios did not differ significantly from the control value of 9.18±2.28 (n=9) (Table 6.1). Percentage reduction in the maximal response of 15.15±2.77% (n=9) in control neurones was not significantly different from 15.69±3.65% (P=0.91, n=7) in depleted neurones and from 12.12±1.94% (P=0.38, n=9) in enriched neurones. The Influence of Membrane Cholesterol on GABA^ Currents page 112 Chapter 6: Ejfect on the Modulation of Ga ABA Currents by Other Drugs Figure 6.6 The effect of membrane cholesterol alterations by methyl-P- cyclodextrin on the antagonism of GABA a currents by 5 |iM bicuculline methochloride. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-P-cyclodextrin (cholesterol depletion) or 0.15 mM cholesterol- methyl-P-cyclodextrin complex (enrichment) for 20 min. (A) Control neurone, (B) Depleted neurone (56% of control), (C) Enriched neurone (182% of control). A. Control neurone GABA(jiM) 1 3 10 30 100 300 1000 3000 + Bicuculline ' (5 n M ) ^ r r ' - f ' r 20 s B. Depleted neurone G A B A (^ M ) 1 3 10 30 100 300 1000 3000 + Bicuculline "V r (5 n M ) T L 20: C. Enriched neurone G A B A (n M ) 1 10 30 100 300 1000 3000 + Bicuculline i r (5 jiM ) T Note: All the control GABA responses were obtained first and those in the presence of drug subsequently. The Influence of Membrane Cholesterol onGABAa Currents page 113 Chapter 6: Ejfect on the Modulation ofGABA^ Currents by Other Drugs Figure 6.7 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on the concentration-response relationship for the antagonism of GABA a currents by 5 pM bicuculline methochloride. Membrane cholesterol was manipulated by incubation at 31"C with 10 mM methyl-p-cyclodextrin (A. cholesterol depletion) or 0.15 mM cholesterol-methyl-p-cyclodextrin complex (B. cholesterol enrichment) for 20 min. A. Cholesterol depletion 0 120 - I 100- < 80- CQ é 60- 40- O Control, GABA alone (n=l A Control, GABA+Bicuculline 20- • Depleted, GABA alone (n=8) 0- A Depleted, GABA+Bicuculline 10'^ 10'® 10'^ 10’^ 10-3 10-2 GABA concentration (M) B. Cholesterol enrichment ^ 100- < 80- CQ S 604 .S 40- O Control, GABA alone (n=8) X A Control, GABA+Bicuculline s 204 • Enriched,GABA alone (n=8) 0- A Enriched,GABA+Bicuculline T 1 I I M i l ) 1---- 1 I I I I I I ) ------1---- T I I I I M [ I---- 1 I I m u ------1----1 I I l l l l | 10'^ 10-6 -10-5 10-4 10-3 10-2 GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 114 Chapter 6: Effect on the Modulation of GABA^ Currents by Other Drugs Figure 6.8 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on the concentration-response relationship for the antagonism of GABA a currents by 10 pM picrotoxinin. Membrane cholesterol was manipulated by incubation at 31°C for 20 min with 10 mM methyl-P-cyclodextrin (A. cholesterol depletion) or 0.15 mM cholesterol-methyl-p-cyclodextrin complex (B. cholesterol enrichment). A. Cholesterol depletion 120n I 100- îi < 8 0 - OQ é 6 0 - 4 0 - Control, GABA alone (n=9) Control, GABA+Picrotoxinin 20- Depleted, GABA alone (n=7) 0- Depleted, GABA+Picrotoxinin I 1 I I iitii| 1 I I m ii| 1 I I iiiii| 1 I I iTiii| 1 I IT mu 1 I I iiiii| 10'^ 10'® 10'^ 10"* 10-3 10‘2 10'^ GABA concentration (M) B. Cholesterol enrichment 120-1 I 100 - < 80 - CQ ê 60 - O Control, GABA alone (n=9) 4 0 - V Control, GABA+Picrotoxinin 20 - • Enriched, GABA alone (n=9) 0- ▼ Enriched, GABA+Picrotoxinin TTT) 10 10 10- 10 10-3 10-2 10'^ GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 115 Chapter 6: Ejfect on the Modulation o f GABA^ Currents by Other Drugs 3) Pregnenolone sulphate The inhibition of GABAa currents by 2 0 pM pregnenolone sulphate is shown in figure 6.9. Application of 20 pM pregnenolone sulphate, in the same U-tube solution as the applied GABA, resulted in both a rightward shift of GABA curve and a reduction in its maximum (Figure 6.10). In control neurones, pregnenolone sulphate reduced the peak amplitudes and also increased the fast decay rate of the peak current as compared with application of GABA alone (Figure 6.9). The effect of pregnenolone was sulphate on the shape of GABA-evoked currents 1 also found with picrotoxinin. However, membrane cholesterol alterations did not affect the change in the shape of GABA-evoked currents by pregnenolone sulphate (Figure 6.9) or picrotoxinin. At cholesterol concentrations from 56% to 182% of control, the GABA EC50 ratios did not differ significantly from the control value of 2.11±0.141 (n=10) (Table 6.1) but the reduction in the maximum of 16.2±1.55% (n=10) in control neurones was significantly less (6.2±2.15%, F< 0 .0 1 , n= 8 ) at the highest cholesterol and significantly greater (32.0±2.54%, P<0.01, n=10) at the lowest cholesterol (Figure 6.11). The Influence of Membrane Cholesterol onGABA a Currents page 116 Chapter 6: Ejfect on the Modulation of GABA^ Currents by Other Drugs Figure 6.9 The effect of membrane cholesterol alterations by methyl-P- cyclodextrin on the antagonism of GABA a currents by 20 |LiM pregnenolone sulphate. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (cholesterol depletion) or 0.15 mM cholesterol-methyl-P- cyclodextrin complex (enrichment) for 20 min. (A) Control neurone, (B) Depleted neurone (56% of control), (C) Enriched neurone (182% of control). A. Control neurone GABA (^M ) 0.3 10 30 100 300 1000 + Pregnenolone " Sulphate(20nM) T T L I L 20 s 20 s B. Depleted neurone GABA (nM ) 0.3 3 10 30 100 300 1000 + Pregnenolone — r~ - y ~ Sulphate(20pM) T r T rr L ÎL 20 s 20 s C. Enriched neurone GABA (pM ) 0.3 10 30 100 300 1000 + Pregnenolone Sulphate (20 pM) 20 s 20 s Note: All the control GABA responses were obtained first and those in the presence of drug subsequently. The Influence of Membrane Cholesterol onGABAa Currents page 117 Chapter 6: Ejfect on the Modulation of GABA ^ Currents by Other Drugs Figure 6.10 The effect of membrane cholesterol alterations by methyl-P- cyclodextrin on the concentration-response relationship for the antagonism of GABA a currents by 20 pM pregnenolone sulphate. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-P-cyclodextrin (A. cholesterol depletion) or 0.15 mM cholesterol-methyl-P-cyclodextrin complex (B. cholesterol enrichment) for 20 min. A. Cholesterol depletion % 120- I 100- < 80- 03 3 60- .1 40- X O Control, GABA alone (n=10) O Control, GABA+Pregnenolone sulphate I 20 - • Depleted.GABA alone (n=10) 0- ♦ Depleted,GABA+Pregnenolone sulphate I------1— I - I - I m i|------1— r—I-I I III] ------1— I I I I 1 1 r|------1— i i i 11 i i | 10-7 10-6 IQ-5 10-4 10-3 GABA concentration (M) B. Cholesterol enrichment 0 120n 100J < BO- 03 S 60- 1 .1X 40- O Control, GABA alone (n=10) □ Control, GABA+Pregnenolone sulphate s 204 • Enriched, GABA alone (n=8) 04 ■ Enriched, GABA+Pregnenolone sulphate I 1— I I I T I i i | 1— I I I I I i r [ 1— I I 1 I I i i | 1— I I I I I i i | 10-7 10-6 10-5 10"* 10-2 GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 118 Chapter 6: Ejfect on the ModulationGABA of a Currents by Other Drugs Figure 6.11 The effect of membrane cholesterol alterations by methyl-p- cyclodextrin on the % reduction of the maximal GABA responses caused by 20 pM pregnenolone sulphate. Membrane cholesterol was manipulated by incubation at 31°C with 10 mM methyl-p-cyclodextrin (depletion) or 0.15 mM cholesterol-methyl- P-cyclodextrin complex (enrichment) for 20 min. 4 0 - * Different from control, /’<0.01, Student's t-test (n=8-10) I 30 20 - 2 10 Depleted Control Enriched Cholesterol levels The Influence of Membrane Cholesterol onGABAa Currents page 119 Chapter 6: Ejfect on the Modulation of GaABA Currents by Other Drugs 6.3 Discussion There was a marked contrast between the influence of membrane cholesterol on the GABA potentiating potency of neuroactive steroids, reported in Chapter 5, and the influence of membrane cholesterol on the GABA potentiating potency of propofol, flunitrazepam and pentobarbitone. Rather than reducing their effects, cholesterol enrichment enhanced the GABA potentiation by propofol, flunitrazepam and pentobarbitone (Table 6.1). Cholesterol depletion did not affect the GABA potentiations by propofol, flunitrazepam and pentobarbitone in contrast to the enhancement of the neuroactive steroid effects. This seemingly general effect of cholesterol on the non-steroidal GABA potentiation may result from alterations in the protein-lipid interface or an allosteric action on the GAB A A receptor protein. It could be related to the reduction in the baseline responsiveness to GABA that occurs with cholesterol enrichment (Chapter 4). The phenomenon may well have extended to the neuroactive steroids but was obscured by the greater additional interaction between cholesterol and the neuroactive steroids that now appears to be rather specific. Interestingly, an analogous influence of membrane cholesterol was seen on the effectiveness of the steroidal antagonist of GABA, pregnenolone sulphate, as was found on the steroidal potentiators of GABA. That is to say, the effect of the pregnenolone sulphate in reducing the maximum of the GABA log.concentration- response curve was inversely related to the membrane cholesterol level. This phenomenon was not seen with either bicuculline or picrotoxinin. Although the potency of bicuculline was, indeed, also reduced by cholesterol enrichment, it was not affected by cholesterol depletion. A reduced potency of both The Influence of Membrane Cholesterol onGABA a Currents page 120 ______Chapter 6: Ejfect on the ModulationGABA o f a Currents by Other Drugs GABA and its competitive antagonist in the presence of excess cholesterol may reflect the same change in receptor properties, a change which is distinct from that due to cholesterol depletion when only GABA potency was reduced (Chapter 4). The GABA inhibition by picrotoxinin was not affected by cholesterol depletion or enrichment (Table 6.1), This may because picrotoxinin acts at an allosteric site within the Cl channel pore of GABA a protein (Xu et ah, 1995) which is far from the sites of action of cholesterol at the transmembrane regions of GABA a protein. In summary, the data presented so far suggest more than one mechanism by which membrane cholesterol influence the pharmacological properties of the GABA a receptor. The details of those mechanisms are speculative at this stage and further work to characterise them is required. The Influence of Membrane Cholesterol on GABA^ Currents page 121 CHAPTER 7 EFFECT OF INCUBATION WITH EPICHOLESTEROL ON THE POTENTIATION OF GABAa c u r r e n t s BY DRUGS The Influence of Membrane Cholesterol on GABA^ Currents page 122 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABA ofa Currents by Drugs 7.1 Introduction In studies on some membrane enzymes that are dependent on membrane cholesterol for their activity, substitution of the cholesterol by some closely related sterols did not restore the enzyme activity. For example, epicholesterol, androstenol, dihydrocholesterol, lanosterol and ergosterol could not restore the activities of the Na"^, K^-ATPase and the Na'^-Ca^'^ exchanger (Vemuri & Phillipson, 1989); cholest- 4-en-3one, 5-androsten-3P-ol-17one, 24ot-methyl-cholest-5-en-3(3-ol, 5a-cholest-24- dien-3(3-ol or 5a-androstan-3p-ol could not restore the activity of GABA transporter (Shouffani & Kanner, 1990). Gimpl et al. (1997), in their studies of the influence of membrane cholesterol on the properties of the oxytocin and cholecystokinin receptors, have extensively explored the consequences of replacing some of the membrane cholesterol with other related sterols. They were able to define rather precise structural requirements for sterols to mimic cholesterol’s influence on the oxytocin receptor, with less stringent requirements with regard to the cholecystokinin receptor. An analogous approach was adopted in the experiments reported in this Chapter but was restricted to the use of epicholesterol, the 3a-hydroxy isomer of cholesterol. Epicholesterol was added to the plasma membrane of control neurones or to substitute cholesterol in cholesterol-depleted neurones by incubation of the neurones with epicholesterol-methyl-|3-cyclodextrin complex. Only a very small amount of tissue was left after dissociation and purification of the neurones and there was no sensitive assay method for the epicholesterol available to us. Therefore, reliance has been placed on the work of Gimpl et al. (1997) showing that epicholesterol incorporates into plasma membranes just as readily as cholesterol from the methyl-(3- cyclodextrin carrier system. The Influence of Membrane Cholesterol onGABA a Currents page 123 Chapter 7: Ejfect o f Incubation with Epicholesterol on the Potentiation oa fCurrents GABA by Drugs The effects of epicholesterol were investigated on the GABA sensitivity of GABA a receptor and the potentiation of GABA a currents by pregnanolone, propofol, flunitrazepam and pentobarbital. The Influence of Membrane Cholesterol onGABA a Currents page 124 Chapter 7: Effect of Incubation with Epicholesterol on the Potentiation ofa Currents GAB A by Drugs 7.2 Experimental protocols and results 7.2.1 Effect of incubation with epicholesterol on the sensitivity of GABAa receptors to GABA Incubation of the neurones with 0.15 mM epicholesterol-methyl-p-cyclodextrin complex at 31°C for 5 and 20 min increased (P<0.0001) the GABA EC 50 value from 5.15±0.14 pM (n=9) in control neurones to 10.55±0.75 pM (n=9) and 15.83±0.56 pM (n=9), respectively (Figure 7.1) with no change in membrane cholesterol. After incubation with 0.15 mM epicholesterol-methyl-P-cyclodextrin complex for 5 and 20 min, the membrane cholesterol contents of the neurones were 1 0 2 ± 1 .6 8 % of control (n=3) and 98.3±2.47% of control (n=3), respectively. The membrane cholesterol content of control neurones was 0.31710.012 pmoles/mg protein (n= 6 ). However, incubation of cholesterol-depleted neurones with 0.1 mM epicholesterol- methyl-p-cyclodextrin complex for 5-20 min could not restore the GABA E C 5 0 (Figure 7.2). The Influence of Membrane Cholesterol onGABA a Currents page 125 Chapter 7: Effect of Incubation with Epicholesterol on the Potentiation ofa CurrentsGABA by Drugs Figure 7.1 The effect of epicholesterol incubation on the GABA concentration- response relationship. The neurones were incubated with 0.15 mM epicholesterol- methyl-p-cyclodextrin complex at 31°C for 5 and 20 min. 00 - 8 0 - 6 0 - 4 0 - Control (n=9) Epicholcsterol for 5 min (n=9) 20 - Epicholesterol for 20 min (n=9) 10- 10-6 10-5 IQ-4 10-= GABA concentration (M) The Influence of Membrane Cholesterol onGABAa Currents page 126 Chapter 7: Effect of Incubation with Epicholesterol on the PotentiationGABA ofa Currents by Drugs Figure 7.2 The effect of incubation of cholesterol-depleted (56% of control) neurones with 0.1 mM epicholesterol-methyl-P-cyclodextrin for 5 and 20 min on the GABA E C 5 0 values. Membrane cholesterol of acutely dissociated hippocampal neurones was depleted by incubation with 10 mM methyl-P-cyclodextrin at 31°C for 20 min. 200 -30 —0— EC 100 N. ★★ III ★* tsP O- * * * *** *** E )-----•------# CU 50-- —0— Cholesterol content (n=3-6) 10 mM MpC 0.1 mM epicholesterol-MpC 0------'------r—^ ------1 0 10 20 30 40 Incubation time (min) nonsignificance, ** ^’<0.005, ***f The Influence of Membrane Cholesterol onGABAa Currents page 127 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABCurrents of by Drugs 7.2.2 Effect of incubation with epicholesterol on the GABA potentiation hy pregnanolone Incubation of the neurones with 0.15 mM epicholesterol-methyl-P-cyclodextrin complex at 31°C for 20 min decreased the GABA EC 50 ratio for 1 |xM pregnanolone from 0.197±0.014 (n=9) in control neurones to 0.151±0.014 (P<0.05, n=8) (Figure 7.3 and Table 7.1). This incubation procedure did not alter the cholesterol content of the neurones (see section 7.2.1). Incubation of neurones depleted of cholesterol to 56% of control with 0.1 mM epicholesterol-methyl-P-cyclodextrin complex for 20 min significantly increased (P<0.01) the GABA EC50 ratio for 1 pM pregnanolone from 0.115±0.011 (n=6) in cholesterol-depleted neurones to 0.162±0.010 (n=8), after 20 min incubation with epicholesterol (Figure 7.4). The latter value was statistically indistinguishable from the GABA EC50 ratio following incubation with epicholesterol in neurones not depleted of their cholesterol. Table 7.1 The effect of incubation with 0.15 mM epicholesterol-methyl-P- cyclodextrin complex for 20 min on the GABA E C 5 0 ratios for the potentiation of drugs. Numbers of experiments are shown in parentheses. GABA EC50 ratio ((GABA+potentiator)/GABA) GABA potentiator Control Epicholesterol-enriched 1 pM Pregnanolone 0.19710.014(9) 0.15110.014* (9) 5 pM Propofol 0.22810.014(10) 0.13110.021** ( 8) 10 pM Flunitrazepam 0.47710.018 (10) 0.29710.010*** ( 8 ) 20 pM Pentobarbital sodium 0.56310.023 ( 8 ) 0.47710.021* ( 8 ) * P<0.05, ** f <0.005, *** P<0.0001, Student’s Mest. The Influence of Membrane Cholesterol GABAon a Currents page 128 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABCurrents of by Drugs Figure 7.3 The effect of incubation of the neurones with 0.15 mM epicholesterol- methyl-P-cyclodextrin inclusion complex for 20 min on the concentration-response relationship for the potentiation of GABA a cuirents by 1 pM pregnanolone. 120- & 100 - < 80- pa é 60- □ Control, GABA alone (n=9) .1 40- X A Control, GABA+Pregnanolone 2 0 - ■ Epicholesterol, GABA alone (n=9) A Epicholesterol, GABA+Pregnanolone 0 - 1-----1 I M I ll|------1-----1 T I t M l)------1— r 10 -7 10-G 10'^ 10" 10-= GABA concentration (M) The Influence of Membrane Cholesterol on GABA^ Currents page 129 Chapter 7: Effect of Incubation with Epicholesterol on the PotentiationGABCurrents of by Drugs Figure 7.4 The effect of incubation of cholesterol-depleted neurones (56% of control) and control neurones with 0.10 and 0.15 mM epicholesterol-methyl-P- cyclodextrin complex (Epich), respectively, for 20 min on the GABA EC 50 ratios for the potentiation of GABA a currents by 1 pM pregnanolone. 0.25n * P<0.05, ** P<0.001, from control, Student t-test NS nonsignificant ly different from control nonsignificantly different from each other 0.204 o MS,# u % 0.154 < ^ 0.10- Ü 0.05- 0.00 Control Control Depleted Depleted + + Epich Epich 20 min 20 min The Influence of Membrane Cholesterol on GABA^ Currents page 130 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABA ofa Currents by Drugs 7.2.3 Effect of incubation of the neurones with epicholesterol on the potentiation of GABAa currents hy other drugs the As found in 1 pregnanolone experiment, the incubation of acutely dissociated hippocampal neurones with 0.15 mM epicholesterol-methyl-P-cyclodextrin inclusion complex at 31°C for 20 min also enhanced the potentiations of GABA a currents by all the potentiators tested, 5 pM propofol, 20 pM pentobarbital sodium and 10 pM flunitrazepam (Figure 7.5 and Table 7.1). The Influence of Membrane Cholesterol onGABA a Currents page 131 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABA ofa Currents by Drugs Figure 7.5 The effect of incubation with 0.15 mM epicholesterol-methyl-(3- cyclodextrin complex for 20 min on the concentration-response relationship for the potentiation of GABA a currents by 5 pM propofol (A), 20 pM pentobarbital sodium (B) and 10 pM flunitrazepam (C). A. 5 )Lim P ro p o fo l I 120-1 100- < CD 80 — < O Control, GABA alone (n=10) Ü 60- ■a E 4 0 - □ Control, GABA+Propofoi S 2 0 - • Epicholesterol incubated, GABA alone (n=8) If 0 - ■ Epicholesterol incubated, GABA+Propofol I—r I ri iiii|—ITU mi| - I I— i~i I i n i i| 10'^ 10-® 10' 10-" 10' GABA concentration (M) B, 20 pm Pentobarbital sodium 120-, §. 2 100- < CO 80 - < / r O Control GABA alone (n=8) o 60 - -a E 4 0 - a y J A Control GABA+20pM Pentobarbital 'i 2 0 - • Epicholesterol, GABA alone (n=8) 0 - Epicholesterol, GABA+20pM Pentobarbital ■ n n p 7 m p TT"r TITIl 10 - 10' 10 - 10 - 10' GABA concentration (M) C. 10 pm Flunitrazepam 1 2 0 -1 1 2 1 0 0 - < CO 8 0 - O O Control, GABA alone (n=10) 13 6 0 - E 4 0 - V Control, GABA+Flunitrazepam 1 Z 2 0 - • Epicholesterol incubated, GABA alone (n=8) (f 0 - ▼ Epicholesterol incubated, GABA+Flunitrazepam itttt—r mn]—r TTT7T, 10'^ 1Q-® 10'^IQ-5 10'^ 10'^ GABA concentration (M) The Influence of Membrane Cholesterol onGABAa Currents page 132 Chapter 7: Ejfect of Incubation with Epicholesterol on the PotentiationGABA ofa Currents by Drugs 7.3 Discussion Gimpl et al. (1997) have shown that epicholesterol can adequately substitute for cholesterol in maintaining membrane fluidity, as measure by 1,6-diphenyl-1,3,5- hexatriene fluorescence anisotropy. In the present experiment, epicholesterol showed some different effects from cholesterol suggesting that mechanisms other than, or in addition to, membrane fluidity changes were responsible for sterol modulation of the GABAa receptor. The fact that incubation of the neurones with 0.15 mM epicholesterol-methyl-(3- cyclodextrin complex increased the GABA EC 50 values with no change in membrane cholesterol indicates that epicholesterol was incorporated into the neurones. There are quite different interpretations of this effect of epicholesterol on GABA E C 5 0 values. The increase in GABA E C 5 0 could be due to epicholesterol mimicking the effect of cholesterol, i.e. equivalent to cholesterol enrichment, or it could be due to epicholesterol having the opposite effect to the endogenous cholesterol, i.e. equivalent to cholesterol depletion. The latter may be the more likely since, in cholesterol- depleted neurones, epicholesterol did not restore the GABA E C 5 0 to the control value, as cholesterol would have done (Figure 4.7). With regard to the potentiation of GABAa currents by drugs in neurones containing normal levels of cholesterol and incubated with epicholesterol-methyl-|3-cyclodextrin complex, the potentiation by all drugs tested was enhanced, including that by pregnanolone (Table 7.1). From Chapter 6 , it is apparent that cholesterol may have two different effects on the action of pregnanolone: ( 1) a general effect, the same as that seen on other potentiators, which is an enhancement of GABA potentiation, (2) a direct interaction specifically with the neuroactive steroid recognition sites which is seen as a reduction in GABA potentiation by pregnanolone. Since cholesterol enrichment reduced the potentiation of pregnanolone, the direct interaction ( 2 ) seems to The Influence of Membrane Cholesterol onGABA a Currents page 133 Chapter 7: Ejfect of Incubation with Epicholesterol on the Potentiation ofGABA^ Currents by Drugs predominate over the general effect (1). In contrast, epicholesterol would appear to act predominantly via general effect (1) in the presence of normal levels of membrane cholesterol. In cholesterol-depleted neurones, substitution of cholesterol with epicholesterol could partially restore the EC50 ratio, for pregnanolone, but longer incubations with the epicholesterol-methyl-p-cyclodextrin were required than with the cholesterol-methyl-p- cyclodextrin for similar degrees of restoration (compare Figure 7.4 with Figure 5.9). Bearing in mind that restoration of the FC 50 ratio equates to a reduction in the potentiating action of pregnanolone, these results suggest that epicholesterol does act via a direct interaction (2) with the neuroactive steroid recognition sites but is less active than cholesterol. The apparently conflicting outcomes of the effects of epicholesterol in (a) the presence of normal membrane cholesterol and (b) in cholesterol-depleted neurones can be resolved if epicholesterol is regarded as a “partial agonist” compared with cholesterol regarded as either an antagonist of pregnanolone at its recognition sites or as full agonist allosteric inhibitor of the action of pregnanolone. Under condition (a), it is unlikely that a partial agonist would add to the existing influence of endogenous cholesterol whereas, under condition (b), a partial agonist might well add to the minimal influence of the depleted endogenous cholesterol. The Influence of Membrane Cholesterol on GABA^ Currents page 134 CHAPTER 8 GENERAL DISCUSSION The Influence of Membrane Cholesterol onGABA a Currents page 135 Chapter 8: General Discussion 8.1 Cholesterol as a modulator of the GABAa receptor Just as many membrane enzymes and receptors including Torpedo nicotinic acetylcholine receptor and human oxytocin and cholecystokinin receptors expressed in HEK-293 cells (Jones & McNamee, 1988; Yeagle, 1991; Gimpl et ah, 1997) have been shown to be influenced by membrane cholesterol, so, in the present study, the GAB A A receptors of acutely dissociated rat hippocampal neurones have also been found to be influenced by membrane cholesterol. Two types of mechanism through which cholesterol may modulate membrane protein function have been proposed (Yeagle, 1993): (1) alteration of the membrane physical properties whereby increased cholesterol reduces the fluidity of the membrane, increases the orientational order of the lipid hydrocarbon chains of membranes and may restrict the free volume available to membrane proteins for conformational changes that may be required for membrane protein function; and (2) direct binding of cholesterol to specific sites on the transmembrane regions of the protein to stimulate or inhibit certain aspects of the function of that protein. In discussing my results on the GABAa receptor, I shall attempt to attribute the observations to one or other of these mechanisms. 8.1.1 The influence of membrane cholesterol on the effect of GABA The GABA-induced Cl' channel opening is composed of three processes (Jones et aL, 1998); (1) GABA binding to its binding site on GABAa receptor; (2) the gating process after GABA binding; and (3) unbinding of the GABA after GABA is washed away. The binding steps determine the fraction of receptors activated, whereas the gating and unbinding steps determine the response duration. Unlike the binding of acetylcholine to nicotinic acetylcholine receptor which is almost as fast as that predicted if the rate-limiting step is the diffusion of ligand into the binding site, the binding and unbinding of GABA to its site depends on receptor structure rather than on diffusion (Jones et aL, 1998). This binding process is an energy-requiring event. The Influence of Membrane Cholesterol onGABA a, Currents page 136 Chapter 8: General Discussion such as a conformational change of the GABA binding site. The binding of two GABA molecules can perform enough work to drive a coupled gating reaction (Jones et aL, 1998). Since increased membrane cholesterol reduced the agonistic effect of GABA (section 4.2.1, Chapter 4), a possible explanation for this result is that cholesterol increased the rigidity of the membrane and, consequently, increased the energy barrier for binding of GABA to result in a reduction of the effect of GABA. The decrease in the competitive antagonistic effect of bicuculline (section 6.2.2, Chapter 6) may support this mechanism. Moreover, the 3a-hydroxyl isomer of cholesterol, epicholesterol, which can change the fluidity of the membrane almost as effectively as cholesterol (Gimpl et aL, 1997), also showed the same effect as cholesterol in lowering the effect of GABA (section 7.2.1, Chapter 7). However, possible effects on the gating process of the Cl' channel should also be considered, since increasing cholesterol from its native level can restrict the free volume for conformational change of the GABAa receptor protein during the gating of Cl' channel, but the coupling of receptor activation to gating probably results in no practical distinction between the two processes. In the case of membrane cholesterol depletion, if it is explained in an analogous way to cholesterol enrichment, i.e. lowering the energy barrier, the effect of GABA should be increased. In fact, the effect of GABA was reduced under this condition (section 4.2.1, Chapter 4). Moreover, the antagonistic effect of bicuculline was not affected (section 6.2.2, Chapter 6). A possible explanation of this is that cholesterol directly interacts with the GABAa receptor protein to augment the ion channel activity in the same way as reported for the nicotinic acetylcholine receptor and some other membrane proteins. For example, cholesterol was found to support the agonist- induced channel opening of nicotinic acetylcholine receptor and stabilise the secondary structure of the receptor’s transmembrane portions (Fong & McNamee, 1987; Femandez-Ballester et aL, 1994). Lack of cholesterol in the membrane resulted in a loss of the ability of this receptor to support cation channel function. In The Influence of Membrane Cholesterol on GABA^ Currents page 137 Chapter 8: General Discussion Other membrane proteins, such as the Na'^, K^-ATPase, the Na'^-Ca^'^ exchanger (Vemuri & Phillipson, 1989), the GABA transporter (Shouffani & Kanner, 1990) and the oxytocin receptor (Gimpl et al., 1997), the requirement for cholesterol to maintain normal function of the protein is highly specific to cholesterol. Likewise, for the GAB A A receptor, I have shown that substitution of cholesterol with its 3 a- hydroxyl isomer, epicholesterol, following cholesterol depletion failed to restore the effect of GABA (section 7.2.1, Chapter 7). In contrast, restoration of cholesterol to the cholesterol-depleted neurones did, as expected, restore the effect of GABA (section 4.2.2, Chapter 4). These results indicate that the P configuration of the 3- hydroxy substituent of cholesterol might be important for the action of cholesterol in supporting GABA-induced Cl" channel opening. 8.1.2 Cholesterol interaction with modulators of the G ABA a receptor Membrane cholesterol enrichment from its native level had a wide-ranging effect of enhancing the potentiation of GABA a currents by propofol, flunitrazepam and pentobarbital (Chapter 6). Presumed enrichment with epicholesterol had a similar effect, which may result from the same mechanism as that which reduced the potency of GABA in cholesterol or epicholesterol enriched neurones. Thus, this general effect on GABA potentiators may be due to alteration of membrane physical properties by cholesterol. As a quite separate effect, cholesterol seems to interact selectively with both positive (pregnanolone and alphaxalone) and negative (pregnenolone sulphate) modulatory neuroactive steroids (Chapter 5 and 6). This effect of cholesterol is a reduction in the activity of the neuroactive steroids which is related to the membrane cholesterol level on a wide range from half to double the normal level of cholesterol. The data do not distinguish between the possibilities (1) that cholesterol is an antagonist of the neuroactive steroids at their recognition sites and (2) that cholesterol is an agonist at The Influence of Membrane Cholesterol on G ABAa Currents page 138 Chapter 8: General Discussion an allosteric site that inhibits the effects of the neuroactive steroids. As argued in the previous chapter, epicholesterol may be regarded as a partial agonist in relation to either of the above concepts of the action of cholesterol, at least in respect of potentiation of GABA by pregnanolone. Altogether, these data support the proposal of a specific protein binding site(s) for cholesterol in its selective interaction with the neuroactive steroids. It remains to be investigated whether the same mechanism underlies the interactions of cholesterol with the GABA-potentiating neuroactive steroids and the GABA- antagonistic pregnenolone sulphate. It is generally assumed that these two classes of steroids have separate sites of action on the GABA a receptor so it is intriguing that cholesterol affects both of them in as apparently similar way. The comparative structural specificities for these effects of cholesterol need to be determined. 8.2 Comparison with [^HJflunitrazepam binding studies The present electrophysiological data are rather difficult to compare with the [^H]flunitrazepam binding data published from this laboratory. The experiments of Bennett & Simmonds (1996) and Schlepper et aL (1998) adopted the conventional 4°C condition for [^H]flunitrazepam binding and, at this temperature, any effects of cholesterol due to change in membrane fluidity would almost certainly differ from the analogous effect at room temperature. Also, the membrane fragments used in these experiments would have exposed both the inner and the outer leaflets of the plasma membrane bilayer to enrichment with cholesterol. The difficulties are further compounded by some lack of reproducibility of result between the two studies. More recent work, briefly reported by Haneef & Simmonds (1999) but largely unpublished, has used room temperature for the binding of [^H]flunitrazepam and, instead of single trace concentrations of [^H]flunitrazepam, has used a range of concentrations that enable estimates of K d and Bmax to be made. On adult rat The Influence of Membrane Cholesterol onGABAa Currents page 139 Chapter 8: General Discussion cerebral cortical membranes enrichment with cholesterol increased the affinity of [^H]flunitrazepam binding compared with that in cholesterol-depleted membranes. This would accord with the increased potentiation of GABA by flunitrazepam in the present study (section 6.2.1, Chapter 6). Enhancement of [^H]flunitrazepam binding by neuroactive steroids was greater in cholesterol-depleted than in cholesterol- enriched membranes, which would appear to accord with the effects of cholesterol on neuroactive steroid potentiation of responses to GABA, except that a similar effect was seen with propofol in the [^H] flunitrazepam binding studies but not in the present electrophysiological studies. This result would seem to indicate some differences between the cholesterol modulations of the pharmacologies of [^H]flunitrazepam binding to adult rat cortical receptors and current responses to GABA on immature rat hippocampal neurones. A further description of these differences must await more complete reports of the [^H]flunitrazepam binding studies. 8.3 Sites of cholesterol-neuroactive steroid interaction The earlier discussion of mechanisms to account for the inverse relationship between neuroactive steroid activity and membrane cholesterol levels offered two possibilities: (1) that cholesterol acts as a competitive antagonist at the neuroactive steroid recognition sites; and (2) that cholesterol binds to a separate site that allosterically modulates the GABA potentiating and antagonistic processes operated by the neuroactive steroids. Further consideration of these two possibilities raises other questions. Where are the recognition sites for the neuroactive steroids located on the GABA a receptor protein and, if there are multiple sites, are they all accessed by the relatively brief exposures to neuroactive steroid employed in the present study? Does the enrichment and depletion of cholesterol affect only the outer leaflet of the plasma membrane bilayer or is the inner leaflet affected as well? The possible sites of cholesterol-neuroactive steroid interaction are shown in figure 8.1. The Influence of Membrane Cholesterol on GABA^ Currents page 140 Chapter 8: General Discussion Figure 8.1 A model (not to scale) for the possible sites of interaction between cholesterol and neuroactive steroids on the GABAa receptor protein. The annular sites are around the outer surface of the protein, while the nonannular sites may be located at the protein/protein interfaces of the subunits. Adapted from Jones & McNamee (1988). lixtraccilular sites Nonaiinular sites Extracellular ^ EXjtei-leaflet I jl I H k Inner leaflet Intracellular Annular sites • •• Annular sites Nonannular site Plan view The Influence of Membrane Cholesterol GABAon a Currents page 141 Chapter 8: General Discussion It seems unlikely that there are recognition sites for the GABA-potentiating neurosteroids on the cytoplasmic domains of the GABA a receptor or, indeed on the transmembrane segments that pass through the inner leaflet of the plasma membrane bilayer, since intracellular application of the neuroactive steroid does not change the normal effect of extracellularly applied neuroactive steroid (Lambert et a l, 1990). This observation would also suggest that the neuroactive steroid does not cross the inner and outer leaflets of the membrane to any significant extent. Evidence that at least some of the effects of pregnanolone are exerted on the transmembrane segment, most likely at the level of the outer leaflet, comes from the unpublished observations of Haneef & Simmonds. Substitution of membrane cholesterol with epicoprostanol (5P-cholestan- 3a-ol), by incubation with methyl-P-cyclodextrin (cholesterol depletion) followed by incubation with epicoprostanol-methyl-P-cyclodextrin complex, enhanced [^H] flunitrazepam binding in rat synaptic membranes Addition of pregnanolone to the binding reaction in these epicoprostanol-enriched membranes, failed to show its usual potentiation of ^H]flunitrazepam. However, when applied acutely in the binding reaction to normal membranes, epicoprostanol failed to enhance [^H]flunitrazepam binding. These results suggest that epicoprostanol has a weak, pregnanolone-like potentiating effect on [^H]flunitrazepam binding that requires substantial loading of the membranes with epicoprostanol to become apparent. The conclusion must be, therefore, that the recognition site for pregnanolone and epicoprostanol is on the transmembrane domain of the GABA a receptor protein. The enrichment and depletion of membrane cholesterol will presumably have a greater effect on the cholesterol content of the outer leaflet compared to the inner leaflet of the plasma membrane due to the extracellular contact with the cholesterol carrier systems. However, the achievement of a 50% depletion in total cholesterol, when there is evidence that the inner (cytofacial) leaflet contains more than 85% of total cholesterol in synaptic plasma membrane of mouse (Igbavbao et al, 1996; Wood et a l, 1999), suggests that the change in membrane cholesterol would have been reflected in the inner leaflet as well. It may be argued, however, that it is the changes in cholesterol level in the outer leaflet The Influence of Membrane Cholesterol on GABA^ Currents page 142 Chapter 8: General Discussion that are the more relevant to the specific interaction with neuroactive steroid modulation of the GABA a receptor. This is based on the observations that the cholesterol analogue epicoprostanol has a weak pregnanolone-like activity (discussed above) which, in turn, favours the notion that cholesterol itself acts as a competitive antagonist at the neuroactive steroid recognition sites. All this strengthens the proposal that important recognition sites for the neuroactive steroids are located on the outer half of the transmembrane segments of the GABA a receptor. It cannot be excluded, however, that a small component of neuroactive steroid activity involves recognition sites on the extracellular domain of the receptor that are not directly influenced by cholesterol. 8.4 Implications of cholesterol interaction with the GABAa receptor If the alterations of membrane cholesterol that were achieved in vitro can be mimicked in vivo, it would have some interesting implications. Much will depend upon whether the levels of plasma membrane cholesterol in neurones of the central nervous system (CNS) are subject to fluctuation in vivo. The turnover of brain cholesterol is very low, with half-time of about 6 months (Liitjohann et aL, 1996), but a substantial proportion of this cholesterol is associated with myelin (Jurevics & Morell, 1995). The blood-brain barrier permits only a very small lipoprotein- mediated flux of cholesterol from the plasma, most of the cholesterol being synthesised within the brain (Liitjohann et aL, 1996). The result of this is that the cerebrospinal fluid levels of cholesterol are some 40-50-fold lower than the blood plasma cholesterol. Nevertheless, those CNS structures in the hypothalamic area that are weakly protected by the blood-brain barrier may be exposed to higher concentrations of cholesterol and may be vulnerable to fluctuations in the plasma cholesterol. Thus, the fluctuations in the circulation levels of cholesterol could influence GABA a receptor-mediated inhibition in certain brain areas. The Influence of Membrane Cholesterol on GABA^ Currents page 143 Chapter 8: General Discussion Cholesterol plays a major role in coronary heart disease. Elevated total cholesterol in the blood stream is correlated with the incidence of coronary heart disease. In the central nervous system, the association between alterations of cholesterol dynamics and pathophysiology is not well understood. Accumulating evidence suggests that naturally low or clinically reduced cholesterol is associated with an increased incidence of suicide and accident which may be mediated by the adverse changes in behaviour and mood (Kaplan et aL, 1997). Low serum cholesterol is also associated with the incidence of depressive illness (Morgan et ah, 1993). Moreover, drugs which lower serum cholesterol, e.g. pravastatin, result in a significant decrease in erythrocyte and platelet membrane cholesterol in hypercholesterolaemic patients (Lijnen et a l, 1996), and could, by analogy, also affect brain functions. A disease in which cholesterol homeostasis is clearly disrupted is Niemann-Pick C disease (NP- C), an inherited lipid disorder due to a mutation in a gene NPC-1 (Wood et aL, 1999). This gene encodes a protein, NP-C protein, which is a putative determinant of intracellular cholesterol transport. This defect causes the accumulation of unesterified cholesterol in the cells leading to cell dysfunction and cell death. Neurological damage in NP-C patients includes visual disorders, movement disorder, dementia and seizures (Yadid et aL, 1998). Alzheimer’s disease (AD) may be a disease in which cholesterol homeostasis is altered. Brain cholesterol content is lower in AD patients than in nondemented subjects (Roth et aL, 1995). The molar ratio of cholesterol: phospholipid decreased 30% in cell membranes. It is not clear how much of this results from loss of myelin but any changes in the plasma membrane composition and structure during aging and AD could alter many membrane proteins, enzymes and receptors such as muscarinic, Di, D 2, ai- adrenergic, az-adrenergic, ^-adrenergic receptors (Roth et aL, 1995), nicotinic acetylcholine receptors (Jones & McNamee, 1988), GABA a receptors (Chapter 4), Na"^, ATPase, Na'^-Ca^'^ exchanger (Yeagle, 1991), the GABA transporter, the Glutamate transporter (Shouffani & Kanner, 1990) and adenylate cyclase (Whetton et The Influence of Membrane Cholesterol onGABA a, Currents page 144 Chapter 8: General Discussion at., 1983). In addition, as shown in this thesis, membrane cholesterol alterations can also affect the actions of many drugs such as neuroactive steroids (Chapter 5), benzodiazepines, barbiturates and propofol (Chapter 6). This could become therapeutically relevant if neuronal membrane cholesterol is subject to fluctuation. 8.5 Future perspectives It has been clear froni this thesis that cholesterol is necessary for the function of GABAa receptor which has also been shown for the nicotinic acetylcholine receptor, a member of the same family of ligand-gated ion channels. The influence of cholesterol on other ligand-gated ion channels such as the strychnine-sensitive glycine receptor, the 5 -HT3 receptor and the AMPA and NMDA receptors, would also be interesting to investigate. Since the mechanisms and the site(s) proposed for the modulation of the GABAa receptor by cholesterol are still rather speculative, a structure-activity analysis with cholesterol replaced by a variety of cholesterol analogues and concomitant biophysical measurements such as fluidity, may provide more evidence of the action of cholesterol. In addition to cholesterol, some other membrane lipids such as phosphatidylserine can also modulate the GABA a receptor (Hammond & Martin, 1987). Therefore, the influence of other membrane lipids on the GABA a receptor should also be investigated to understand the whole picture of the influence of membrane constituents on the function of the GABA a receptor. This might be explored by reconstitution of solubilised GABA a receptors into liposomes of known lipid composition for ion efflux or receptor binding studies. The Influence of Membrane Cholesterol onGABAa Currents page 145 Acknowledgements I would like to express my thanks to the following: My supervisor Professor Michael A. Simmonds for all his help, guidance and encouragement during my research and writing of this thesis. Professor T.G. Smart, Dr. A. Amato and Dr. P. Thomas for helping with the patch clamp technique. Cris Anizeski and Yu Zhong for helping me in the initial stage. All my friends in the department for helping me in many different ways. All my Thai friends in the School of Pharmacy, Ruedeekom Wiwattanapatapee, Surassawadee Suntharasaj, Jittima Chatchawalsaisin, Parinya Arunothayanun and Sasitom Kittivoravitkul, for their help and support. My colleagues in the Faculty of Pharmaceutical Sciences, Chulalongkom university, Thailand, especially Prof. Pavich Tongroach and Prof. Sumlee Jaidee, for their help, support and encouragement. Chulalongkom University, Thailand for financial support. Finally, I would like to thank my mother for her love and support. The Influence of Membrane Cholesterol on GABA^ Currents page 146 REFERENCES The Influence of Membrane Cholesterol on GABA^ Currents page 147 References Addona G.H., Sandermann Jr. H., Kloczewiak M.A., Hussain S.S. and Miller K.W. (1998) Where does cholesterol act during activation of the nicotinic acetylcholine receptor? Biochim. Biophys. Acta, 1370: 299-309. Allain C.C., Poon L.S., Chan C.S., Richmond W. and Fu P C. (1974) Enzymatic determination of total serum cholesterol. Clin. Chem., 20:470-475 Barnard E.A., Skolnick P., Olsen R.W., Mohler H., Sieghart W., Biggio G., Braestrup C., Bateson A.N. and Langer S.Z. (1998) International Union of Pharmacology. XV. Subtypes of y-aminobutyric acid receptors: Classification on the basis of subunit structure and receptor function. Pharmacol Rev., 50: 291-313. Baulieu E.E. and Robel P. (1998) Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive neurosteroids. Proc. Natl Acad. Scl USA, 95: 4089-4091. Bennett P.J. and Simmonds M.A. (1996) The influence of membrane cholesterol on GABAa receptor. Br. J. Pharmacol, 117: 87-92. Bittman R. (1993) A review of the kinetics of cholesterol movement between donor and acceptor bilayer membranes. In: Cholesterol in membrane models (Finegold L., Ed.), pp. 45-65. CRC Press, Boca Raton. Boss B.D., Gozes I. and Cowan W.M. (1987) The survival of dentate gyrus neurons in dissociated culture. Dev. Brain Res., 36: 199-218. Bowery N.G. (1993) GAB As receptor pharmacology. Annu. Rev. Pharmacol. Toxicol, 33: 109-147. The Influence of Membrane Cholesterol onGABA a Currents page 148 ______References Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 72: 248-254. Bristow D.R. and Martin I.L. (1987) Solubilisation of the y-aminobutyric acid/benzodiazepine receptor from rat cerebellum: Optimal preservation of the modulatory responses by natural brain lipids. J. Neurochem., 49:1386-1393. Castuma C.E. and Brenner R.R. (1986) Cholesterol-dependent modification of microsomal dynamics and UDP glucuronyltransferase kinetics. Biochemistry, 25: 4733-4738. Chen G., Trombley P.Q. and van den Pol A.N. (1996) Excitatory actions of GABA in developing rat hypothalamic neurones. J, Physiol, 494: 451-464. Corbin J., Wang H.H. and Blanton M.P. (1998) Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [ ^ azido-cholesterol. Biochim. Biophys. Acta, 1414: 65-74. Cotman C., Blank M.L., Moehl A. and Snyder F. (1969) Lipid composition of synaptic plasma membrane isolated from rat brain by zonal centrifugation. Biochemistry, 8: 4606-4612. Cottrell G.A., Lambert J.J. and Peters J.A. (1987) Modulation of GABAa receptor activity by alphaxalone. Br. J. Pharmacol, 90: 491-500. Crain R.C. and Ziversmit D.B. (1980) Two-nonspecific phospholipid exchange proteins from beef liver. 1. Purification and characterization. Biochemistry, 19: 1433-1439. The Influence of Membrane Cholesterol on GABA^ Currents page 149 References Crews F.T., Camacho A. and Phillips M.L (1983) Cholinergic stimulation of hippocampal pyramidal cells is inhibited by increasing membrane cholesterol. Brain Res., 261: 155-158. Davidson W.S., Rodrigueza W.V., Lund-Katz S., Johnson W.J., Rothblat G.H. and Phillips M.C. (1995) Effects of acceptor particle size on the efflux of cellular free cholesterol. J. Biol. Chem., 270: 17106-17113. Davies P.A., Hanna M.C., Hales T.G., and Kirkness E.F. (1997) Insensitivity to anaesthetic agents conferred by a class of GABAa receptor subunit. Nature, 385: 820- 823. Demel R.A., Bruckdorfer K.R. and van Deenen L.L.M. (1972) The effect of sterol structure on the permeability of liposomes to glucose, glycerol and Rb^. Biochim. Biophys.Acta, 255: 321-330. Fenwick E.M., Marty A. and Neher E. (1982) A patch-clamp studies of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physio., 331: 577-597. Femandez-Ballester G., Castresana J., Fernandez A.M., Arrondo J.L.R., Ferragut J.A. and Gonzalez-Ros J.M. (1994) A role for cholesterol as a structural effector of the nicotinic acetylcholine receptor. Biochemistry, 33:4065-4071. ffrench-Mullen J.M.H. and Spence K.T. (1991) Neurosteroids block Ca^"^ channel current in freshly isolated hippocampal CAl neurons. Eur. J. Pharmacol., 202: 269- 272. The Influence of Membrane Cholesterol on GABA^ Currents page 150 References Fong T.M. and McNamee M.G. (1986) Correlation between acetylcholine receptor function and structural properties of membranes. Biochemistry, 25: 830-840. Gasior M., Carter R.b. and Witkin J.M. (1999) Neuroactive steroids: potential therapeutic use in neurological and psychiatric disorders. Trends. Pharmacol. Sci., 20: 107-112. Gimpl G., Burger K. and Fahrenholz F. (1997) Cholesterol as modulator of receptor function. Biochemistry, 36: 10959-10974. Hamil O.P., Marty A., Neher E., Sakmann B. and Sigworth F.J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluger Arch., 391:85-100. Hammond J.R. and Martin I.L. (1987) Modulation of [^HJflunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine. Eur. J. Pharmacol., 137: 49-58. Haneef F. and Simmonds M.A. (1999) Influence of membrane cholesterol on flunitrazepam binding and its modulation by pregnanolone and propofol. J. Physiol., 518: 160P. Kara M., Kai Y. and Ikemoto Y. (1993) Propofol activates GABAa receptor-chloride ionophore complex in dissociated hippocampal pyramidal neurones of the rat. Anesthesiology, 79: 781-787. Harrison N.L., Majewska M.D., Harrington J.W. and Barker J.L. (1987) Structure- activity relationships for steroid interaction with the y-aminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther., 241: 346-353. The Influence of Membrane Cholesterol on GABA^ Currents page 151 References Harrison N.L. and Simmonds M.A. (1984) Modulation of the GAB A receptor complex by a steroid anaesthetic. Brain Res,, 323: 287-292. Hawkinson I.E., Acosta-Burruel M., Yang K.C., Hogenkamp D.J., Chen J.S., Lan N.C., Drewe J.A., Whittemore E.R., Woodward R.M., Carter R.B. and Upasani R.B. (1998) Substituted 3P-phenylethynyl derivatives of 3a-hydroxy-5a-pregnan-20-one: Remarkably potent neuroactive steroid modulators of y-aminobutyric acidA receptors. J. Pharmacol. Exp. Ther., 287: 198-207. Hedblom E. and Kirkness E.F. (1997) A novel class of GABAa receptor subunit in tissues of the reproductive system. J. Biol. Chem., 272: 15346-15350. Igbavboa U., Avdulov N.A., Schroeder F. and Wood W.G. (1996) Increasing age alters transbilayer fluidity and cholesterol asymmetry in synaptic plasma membranes of mice. J. Neurochem., 66: 1717-1725. Jefferson J R., Slotte J.P., Nemecz G., Pasuszyn A., Scallen T.J. and Schroeder F. (1991) Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein. J. Biol. Chem., 266: 5486-5496. Jones O.T. and McNamee M.G. (1988) Annular and nonannular binding sites for cholesterol associated with the nAChR. Biochemistry, 27: 2364-2374. Jones M.V., Sahara Y., Dzubay J.A. and Westbrook G.L. (1998) Defining affinity with the GABAa receptor. / Neurosci., 18: 8590-8604. Jurevics H. and Morell P. (1995) Cholesterol for synthesis of myelin is made locally, not imported into brain. J. Neurochem., 64: 895-901. The Influence of Membrane Cholesterol on G ABAa, Currents page 152 References Kaneda M,, Nakamura H. and Akaike N. (1988) Mechanical and enzymatic isolation of mammalian CNS neurons. Neuroscience Res., 5:299-315. Kaplan J.R., Muldoon M.F., Manuck S.B. and Mann J.J. (1997) Assessing the observed relationship between low cholesterol and violence-related mortality. Implications for suicide risk. Annals of the New York Academy of Science. 836: 57-80. Kilsdonk E.P.C., Yancey P.O., Stoudt G.W., Bangerter F.W., Johnson W.J., Phillips M.C. and Rothblat G.H. (1995) Cellular cholesterol efflux mediated by cyclodextrin. J. Biol. Chem., 270: 17250-17256. Klein U., Gimpl G. and Fahrenholz F. (1995) Alteration of the myométrial plasma membrane cholesterol content with |3-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry, 34: 13784-13793. Lambert J.J., Belleli D., Hill-Venning C. and Peters J.A. (1995) Neurosteroids and GAB Aa receptor function. Trends Pharmacol. Sci., 16:295-303. Lambert J.J., Peters J.A., Sturgess N.C. and Hales T.G. (1990) Steroid modulation of the GABAa receptor complex: electrophysiological studies. In Steroid and neuronal activity (Ciba Foundation Symposium 153), pp 56-82, Wiley, Chichester. Lijnen P. and Petrov V. (1995) Cholesterol modulation of transmembrane cation transport systems in human erythrocytes. Biochem. Mol. Medicine, 56: 52-62. Liscum L. and Underwood K.W. (1995) Intracellular cholesterol transport and compartmentation. J. Biol. Chem., 270: 15443-15446. The Influence of Membrane Cholesterol onGABA a Currents page 153 References Lütjohann D., Breuer O., Ahlborg G., Nennesmo L, Sidén Â., Diczfalusy U. and Bjôrkhem L (1996) Cholesterol homeostasis in human brain: Evidence from an age- dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc. Natl Acad. Sci. USA, 93: 9799-9804. Macdonald R.L. and Olsen R.W. (1994) GABAa receptor channels. Annu. Rev. Neurosci., 17: 569-602. Majewska M.D. (1992) Neurosteroids : Endogenous bimodal modulators of the GABAa receptor, Mechanism of action and physiological significance. Prog. NeurobioL, 38: 379-395. Majewska M.D., Harrison N.L., Schwartz R.D., Barker J.L. and Paul S.M. (1986) Steroid hormone metabolites are barbiturate like modulators of the GABAa receptor. Science, 232: 1004-1007. Majewska M.D. and Schwartz R.D. (1987) Pregnanolone sulfate: an endogenous antagonist of the y-aminobutylic acid receptor complex in brain? Brain Res., 404: 355-360. McKeman R.M. and Whiting P.J. (1996) Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci., 19: 139-143. Mensah-Nyagan A.G., Do-rego J.L., Beaujean D., Luu-The V., Pelletier G. and Vaudry H. (1999). Neurosteroids: Expression of steroidogenic enzymes and regulation of steroid biosynthesis in the central nervous system. Pharmacol Rev., 51: 63-81. The Influence of Membrane Cholesterol onGABAa Currents page 154 References Mitchell D.C., Straume M., Miller J.L. and Litman B.J. (1990) Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids. Biochemistry, 29: 9143-9149. Morgan R.E., Palinkas L.A., Barrett Connor E.L. and Wingard D.L. (1993) Plasma cholesterol and depressive symptoms in older man. Lancet, 341: 8837, 75-79. Mouritsen O.G. and Biltonen R.L. (1993) Protein-lipid interactions and membrane heterogeneity. In New comprehensive biochemistry (Volume 25) : Protein-lipid interactions (Watts A., Ed), pp. 1-39, Elsevier, Amsterdam. Miitsch B., Gain s N. and Hauser H. (1986) Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact. Biochemistry, 25: 2134- 3140. Nayeem N., Green T.P., Martin I.L. and Barnard E.A. (1994) Quaternary structure of the native GABAaR determined by electron microscope image analysis. J. Neurochem., 62: 815-818. New R.R.C. (1990) Introduction, In Liposomes : A practical approach (New, R.R.C., Ed), pp. 1-32, Oxford University Press, New York. Ohtani Y., Irie T., Uekama K., Fukunaga K. and Pitha J. (1989) Differential effects of alpha-, beta- and gamma-cyclodextrin on human erythrocytes. Eur. J. Biochem., 186:17-22. The Influence of Membrane Cholesterol onGABA a Currents page 155 References Park-Chung M., Malayev A.A., Purdy R.H., Gibbs T.T. and Farb D.H. (1999) Sulfated and unsulfated steroids modulate gamma-aminobutyric acid A receptor function through distinct sites. Brain Res., 830: 72-87. Park-Chung M., Wu F., Purdy R.H., Malayev A.A., Gibbs T.T. and Farb D.H. (1997) Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids. Mol. Pharmacol., 52: 1113-1123. Paul S.M. and Purdy R.H. (1992) Neuroactive steroids. FASEB J., 6: 2311-2322. Peters J.A., Kirkness E.F., Callachan H., Lambert J.J. and Turner A.J. (1988) Modulation of GABAa receptor by depressant barbiturates and pregnane steroids. Br. J. Pharmacol., 94: 1257-1269. Pitha J., Irie T., Sklar P.B. and Nye J.S. (1988) Drug solubilizers to aid pharmacologist: amorphous cyclodextrin derivatives. Life Sci., 43:493-502. Prince R.J. and Simmonds M.A. (1992) Propofol potentiates the binding of flunitrazepam to the GABAa receptor complex. Brain Res., 596: 238-242. Rodrigueza W.V., Wheeler J.J., Klimuk S.K., Kitson C.N. and Hope M.J. (1995) Transbilayer movement and net flux of cholesterol and cholesterol sulfate between liposomal membranes. Biochemistry, 34: 6208-6217. Roth G.S., Joseph J.A. and Mason R.P. (1995) Membrane alterations as causes of impaired signal transduction in Alzheimer’s disease and aging. Trends Neurosci., 18: 203-206. The Influence of Membrane Cholesterol onGABA a Currents page 156 References Ryan S.E., Demers C.N., Chew J.P. and Baenziger I.E. (1996) Structural effects of neutral and anionic lipids on the nicotinic acetylcholine receptor. J. Biol Chem., 271: 24590-24597. Sakmann B., Hamil O.P. and Bormann J. (1983) Patch-clamp measurements of elementary chloride currents activated by the putative inhibitory transmitters G ABA and glycine in mammalian spinal neurones. J Neural Transm. SuppL, 18: 83-95. Schleper O., Goetgheluck Q. and Simmonds M.A. (1998) Modulation of [^H]- flunitrazepam binding by neurosteroids and cholesterol. Br. J. Pharmacol, 123: 202P. Schroeder P., Jefferson J.R., Kier A.B., Knittel J.,Scallen T.J., Wood W.G. and Hapala I. (1991) Membrane cholesterol dynamics: Cholesterol domains and kinetic pool. Proc. Soc. Exp. Biol Med., 196: 235-252. Shingai R., Yanagi K., Fukushima T., Sakata K. and Ogurusu T. (1996) Functional expression of GAB A rho3 receptors in Xenopus oocytes. Neuroscience Res., 26: 387- 390. Shouffani A. and Kanner B.I. (1990) Cholesterol is required for the reconstitution of the sodium- and chloride-couple, y-aminobutyric acid transporter from rat brain. J. BiolChem., 265: 6002-6008. Sieghart W. (1995) Structure and pharmacology of y-aminobutyric acidA receptor subtypes. Pharmacol Rev., 47: 181-234. Simmonds M.A. (1991) Modulation of the GABA a receptor by steroids. Semin. Neurosci., 3: 231-239. The Influence of Membrane Cholesterol onGABA a Currents page 157 References Singer SJ. and Nicholson G.L. (1972) The fluid mosaic model of the structure of cell membranes. Science, 175: 720-731. Sooksawate T. and Simmonds M.A. (1998) Increased membrane cholesterol reduces the potentiation of GABAa currents by neurosteroids in dissociated hippocampal neurones. Neuropharmacology, 37: 1103-1110. Steck T.L., Kezdy F.J. and Lange Y. (1988) An activation-collision mechanism for cholesterol transfer between membranes. J. Biol. Chem., 263: 13023-13031. Study R.E. and Barker J.L. (1981) Diazepam and (+/-)pentobarbital : Fluctuation analysis reveals different mechanisms for potentiation of y-aminobutylic acid responses in cultured central neurons. Proc. Natl. Acad. Sci. USA, 78: 7180-7184. Sunshine C. and McNamee M.G. (1994) Lipid modulation of nicotinic acetylcholine receptor function: the role of membrane lipid composition and fluidity. Biochim. Biophys. Acta, 1191: 59-64. Twyman R.E., Green R.M. and Macdonald R.L. (1991) Kinetics of open channel block by penicillin of single GABAa receptor channels from mouse spinal cord neurones in culture. J. Physiol., 445: 97-127. Twyman R.E. and Macdonald R.L. (1992) Neurosteroid regulation of GABAa receptor single channel kinetic properties. J. Physiol., 456: 215-245. Valera S., Ballivet M. and Bertrand D. (1992) Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA, 89: 9949-9953. The Influence of Membrane Cholesterol on GABA^ Currents page 158 ______References Vemuri R. and Phillipson K.D. (1989) Influence of sterols and phopholipids on sarcolemma and sarcoplasmic reticular cation transporters. J. Biol Chem., 264: 8680- 8688. Whetton A.D., Gorden L.M. and Housley M.D. (1983) Adenylate cyclase is inhibited upon depletion of plasma membrane cholesterol. Biochem. J., 212: 331-338. Whiting P.J., McKeman R.M. and Wafford K.A. (1995) Stmcture and pharmacology of vertebrate GABAa receptor subtype. Internat. Rev. NeurobioL, 38: 95-138. Wood W.G., Schroeder F., Avdulov N.A., Chochina S.V. and Igbavboa U. (1999) Recent advances in brain cholesterol dynamic: Transport, domain, and Alzheimer’s disease. Lipids, 34: 225-234. Xu M., Covey D.F. and Akabas M.H. (1995) Interaction of picrotoxin with GABAa receptor channel-lining residues probed in cysteine mutants. Biophys. J., 69: 1858- 1867. Yadid G., Sotnik Barkai I., Tomatore C., Barker Cains B., Harvey White J., Pentchev P.G. and Goldin E. (1998) Neurochemical alterations in the cerebellum of a murine model of Niemann-Pick type C disease. Brain Res., 799: 250-256. Yancey P.G., Rodriguenza W.V., Kilsdonk E.P.C., Stoudt G.W. Johnson W.J., Phillips M.C. and Rothblat G.H. (1996) Cellular cholesterol efflux mediated by cyclodextrins. J. Biol Chem., 271: 16026-16034. Yeagle P.L. (1985) Cholesterol and the cell membrane. Biochim. Biophys. Acta, 822: 267-287. The Influence of Membrane Cholesterol onGABA a Currents page 159 References Yeagle P.L. (1991) Modulation of membrane function by cholesterol. Biochemie, 73: 1303-1310. Yeagle P.L. (1993) The biophysics and cell biology of cholesterol: an hypothesis for the essential role of cholesterol in mammalian cells. In Cholesterol in membrane models (Finegold L., Ed), pp. 1-12. CRC Press, Boca Raton. Yeagle P.L., Young J. and Rice D. (1988) Effects of cholesterol on (Na'^,K'^)-ATPase ATP hydrolyzing activity in bovine kidney. Biochemistry, 27: 6449-6452. Zorumski C F., Wittmer L.L., Isenberg K.E., Hu Y. and Covey D.F. (1996) Effect of neurosteroid and Benz[g]indene enantiomers on GABAa receptors in cultured hippocampal neurons and transfected HEK-293 cells. Neuropharmacology, 35: 1161-1168. The Influence of Membrane Cholesterol onGABAa Currents page 160