Potentiation of GABA A receptors by neurosteroids: Mechanisms and sites (original) (raw)

Neuroactive steroids have multiple actions to potentiate GABA A receptors

The Journal of Physiology, 2004

The effects of neuroactive steroids on the function of GABA A receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with α1 β2 γ2L subunits. Activity was elicited with a half-maximal (50 µM) concentration of GABA. Two steroids were studied in detail: ACN ((3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3α,5β,17β)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration-dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC 50 about 180 nM ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC 50 about 35 nM ACN). B285 also increased the prevalence of the longest duration open times (EC 50 about 18 nM B285) but increased the duration only at concentrations close to 10 µM. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC 50 of about 50 nM ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC 50 concentrations (about 40 nM ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 µM ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the γ2 subunit, but not the α1 or β2 subunits, abolished the ability of ACN to increase the duration of OT 3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABA A receptor.

Neurosteroid modulation of GABAA receptors

Progress in Neurobiology, 2003

Certain metabolites of progesterone and deoxycorticosterone are established as potent and selective positive allosteric modulators of the ␥-aminobutyric acid type A (GABA A ) receptor. Upon administration these steroids exhibit clear behavioural effects that include anxiolysis, sedation and analgesia, they are anticonvulsant and at high doses induce a state of general anaesthesia, a profile consistent with an action to enhance neuronal inhibition. Physiologically, peripherally synthesised pregnane steroids derived from endocrine glands such as the adrenals and ovaries function as hormones by crossing the blood brain barrier to influence neuronal signalling. However, the demonstration that certain neurons and glial cells within the central nervous system (CNS) can synthesize these steroids either de novo, or from peripherally derived progesterone, has led to the proposal that these steroids (neurosteroids) can additionally function in a paracrine manner, to locally influence GABAergic transmission. Steroid levels are known to change dynamically, for example in stress and during pregnancy. Given that GABA A receptors are ubiquitously expressed throughout the central nervous system, such changes in steroid levels would be predicted to cause a global enhancement of inhibitory neurotransmission throughout the brain, a scenario that would seem incompatible with a physiological role as a selective neuromodulator. Here, we will review emerging evidence that the GABA-modulatory actions of the pregnane steroids are highly selective, with their actions being brain region and indeed neuron dependent. Furthermore, the sensitivity of GABA A receptors is not static but can dynamically change. The molecular mechanisms underpinning this neuronal specificity will be discussed with particular emphasis being given to the role of GABA A receptor isoforms, protein phosphorylation and local steroid metabolism and synthesis.

Kinetic and Structural Determinants for GABA-A Receptor Potentiation by Neuroactive Steroids

Current Neuropharmacology, 2010

Endogenous neurosteroids and synthetic neuroactive steroid analogs are among the most potent and efficacious potentiators of the mammalian GABA-A receptor. The compounds interact with one or more sites on the receptor leading to an increase in the channel open probability through a set of changes in the open and closed time distributions. The endogenous neurosteroid allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest-lived open time component and by reducing the prevalence of the longest-lived intracluster closed time component. Thus the channel mean open time is increased and the mean closed time duration is decreased, resulting in potentiation of channel function.

Neurosteroid Access to the GABAA Receptor

Journal of Neuroscience, 2005

GABA A receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA A receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABA A receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Auto-modulation of neuroactive steroids on GABAA receptors: A novel pharmacological effect

Neuropharmacology, 2007

GABA A receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA A receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (a1b3d, a1b3g2L, a6b3d, a6b3g2L) and at GABA A receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5a-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at a1b3g2L, a6b3g2L, and a6b3d subtypes while it enhanced potentiation at a1b3d isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA A receptor sites containing the d subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.

Neurosteroids and GABAA receptor function

Trends in Pharmacological Sciences, 1995

In 1994, a potent and selective interaction of the steroidal anaesthetic alphaxalone with the GABA, receptor was demonstrated. Subsequent studies established that certain naturally occurring steroids were potent positive allosteric modulators of the GABA* receptor. Although peripheral endocrine glands are an important endogenous source, the brain can synthesize 'neurosteroids', and these have the potential to influence the activity of the GAB% receptor in the CNS. Systemic administration of steroids have clear behavioural effects. In this article, Jeremy lambert and colleagues review recent advances in this field and discuss the therapeutic potential of this novel, non-genomic effect of steroids and investigate whether they may influence behaviour under physiological, or pathophysiological, conditions. Dundee. Dundee, UK DO1 9% 0 1995,

Mechanisms of neurosteroid interactions with GABAA receptors

Pharmacology & Therapeutics, 2007

Neuroactive steroids have some of their most potent actions by augmenting the function of GABA A receptors. Endogenous steroid actions on GABA A receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA A receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.