Dual Potentiating and Inhibitory Actions of a Benz(e)indene Neurosteroid Analog on Recombinant 122 GABAA Receptors (original) (raw)
Related papers
Molecular Pharmacology, 2006
Benz [e]indenes are tricyclic analogues of neuroactive steroids and can be modulators of GABA A receptor activity. We have examined the mechanisms of action of a benz[e]indene compound [3S-(3α,3aα,5aβ,7β,9aα,9bβ)]-dodecahydro-7-(2hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch clamp and whole-cell recordings from HEK cells transfected with rat GABA A receptor α1, β2, γ2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA A receptor activity with a peak effect at 2 µM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 appears to be mediated through interactions with the classical neuroactive steroid binding site.
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.
Potentiation of GABA A receptors by neurosteroids: Mechanisms and sites
International Congress Series, 2005
Neurosteroids increase the strength of neuronal inhibition by enhancing the activity of GABA-A receptors. The response to lower concentrations of GABA is increased in the presence of these drugs. One question is the nature of the basis for the potentiation — is there a change in the affinity of the receptor for GABA or an increase in the efficacy of activation? The answer for these drugs is that the major change occurs in the efficacy of activation, with no change in channel opening but a decrease in rates for entering states with a closed channel. There are at least 2 sites of action, which can be distinguished by the activity of steroid analogues with different structures. There are at least 3 mechanisms of action. Mutations of GABAA receptor subunits indicate that at least one site for steroid action is likely to be at the extreme carboxyl end of the γ2 subunit. Analysis of data obtained at a low concentration of GABA suggests that the ability of steroids to potentiate the GABAA ...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
Fast applications of GABA (1 mM) to nucleated and outside-out patches excised from granule neurons in cerebellar slices from developing rats evoked currents with a double exponential time course reminiscent of that of IPSCs. A neurosteroid 3␣, 21dihydroxy-5␣-pregnan-20-one (THDOC) remarkably increased the slow deactivation time constant and slowed down recovery from desensitization, as estimated by paired-pulse GABA applications. THDOC also reduced the amplitude of GABA currents, whereas it failed to affect the fast deactivation component and its relative contribution to peak amplitude. The effects of THDOC on slow deactivation were greater in rats younger than postnatal day 13 (P13) as compared with rats at P30-P35. THDOC failed to alter deactivation of short responses induced by a less-potent agonist taurine at saturating doses. These responses had deactivation kinetics described by a fast single exponential decay, little desensitization, and quick recovery. However, THDOC slowed deactivation if taurine responses were long enough to allow consistent desensitization, suggesting that desensitized states are required for the neurosteroid to modulate GABA responses. In outside-out patches, just as desensitized states prolonged GABA responses by producing reopening of channels activated by brief GABA pulses, THDOC increased the channel open probability by further increasing the number of late channel openings, resulting in a prolongation of the slow deactivation. Our data suggest that neurosteroid potentiates the inhibitory postsynaptic transmission via the prolongation of the slow deactivation and that the alteration of kinetics of entry and exit from desensitized states underlies the allosteric modification of GABA A receptors by neurosteroids.
European Journal of Pharmacology, 2007
3β-hydroxysteroids are pregnenolone sulfate-like GABA A receptor antagonists. The aim of the current study was to compare the functional differences between 3β-hydroxysteroids and pregnenolone sulfate to inhibit GABA A receptors expressed in Xenopus oocytes. Recombinant rat GABA A receptors encoding wild type α 1 β 2 γ 2L receptor, mutant α 1V256S β 2 γ 2L and α 1 β 2A252S γ 2L receptors were examined using a two-electrode voltage-clamp technique. A homologous mutation of the residue at 2′position closest to the cytoplasmic end of the M 2 helix to serine on both α 1 and β 2 subunit, α 1V256S and β 2A252S , reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly in the α 1V256S β 2 γ 2L receptor (P b 0.05), whereas it decreased moderately in the α 1 β 2A252S γ 2L receptor. We found that 5α-pregnan-3β, 20(S)-diol (UC1019) and 5β-pregnan-3β, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3β-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC 50 s of 0.20 ± 0.07 μM; 1.88 ± 0.32 μM and 2.58 ± 0.58 μM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants α 1V256S β 2 γ 2L and α 1 β 2A252S γ 2L receptors (P b 0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant α 1V256S β 2 γ 2L receptor. Pregnenolone sulfate promoted slow desensitization with prolonged GABA application in a dose-dependent manner in the wild type receptor, but not mutant receptors. On the contrary, UC1019 and UC1020 (≤20 μM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABA A receptor inhibition by pregnenolone sulfate, but not 3β-hydroxysteroids, was dependent on desensitization kinetics of the Clchannels. A point mutation at M 2 helix of the β 2 -subunit (β 2A252S ) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABA A receptors without affecting the inhibitory properties of 3β-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3β-hydroxysteroids the coincident channel property at the GABA A receptor.
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.
Neurosteroid prolongs GABAA channel deactivation by altering kinetics of desensitized states
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
Fast applications of GABA (1 mM) to nucleated and outside-out patches excised from granule neurons in cerebellar slices from developing rats evoked currents with a double exponential time course reminiscent of that of IPSCs. A neurosteroid 3alpha, 21dihydroxy-5alpha-pregnan-20-one (THDOC) remarkably increased the slow deactivation time constant and slowed down recovery from desensitization, as estimated by paired-pulse GABA applications. THDOC also reduced the amplitude of GABA currents, whereas it failed to affect the fast deactivation component and its relative contribution to peak amplitude. The effects of THDOC on slow deactivation were greater in rats younger than postnatal day 13 (P13) as compared with rats at P30-P35. THDOC failed to alter deactivation of short responses induced by a less-potent agonist taurine at saturating doses. These responses had deactivation kinetics described by a fast single exponential decay, little desensitization, and quick recovery. However, THDOC...
Characterization of neurosteroid effects on hyperpolarizing current at α4β2δ GABAA receptors
Psychopharmacology, 2014
Rationale-The neurosteroid 3α,5β-THP (3α-OH-5β-pregnan-20-one, pregnanolone) is a modulator of the GABA A receptor (GABAR), with α4β2δ GABARs the most sensitive. However, the effects of 3α,5β-THP at α4β2δ are polarity-dependent: 3α,5β-THP potentiates depolarizing current, as has been widely reported, but decreases hyperpolarizing current by accelerating desensitization. Objectives-The present study further characterized 3α,5β-THP inhibition of hyperpolarizing current at this receptor and compared effects of other related steroids at α4β2δ GABARs. Methods-α4β2δ GABARs were expressed in HEK-293 cells, and agonist-gated current recorded with whole cell voltage-clamp techniques using a theta tube to rapidly apply agonist before and after application of neurosteroids. Results-The GABA-modulatory steroids (30 nM) 3α,5α-THP (3α-OH-5α-pregnan-20-one, allopregnanolone) and THDOC (3α,21-dihydroxy-5α-pregnan-20-one) inhibited hyperpolarizing GABA(10 µM)-gated current at α4β2δ GABARs similar to 3α,5β-THP, while the inactive 3β,5β-THP isomer had no effect. Greater inhibition was seen for current gated by the high efficacy agonist gaboxadol (THIP, 100 µM) than for GABA (0.1-1000 µM), consistent with an effect of 3α,5β-THP on desensitization. Inhibitory effects of the steroid were not seen under low [Cl − ] conditions or in the presence of calphostin C (500 nM), an inhibitor of protein kinase C. Chimeras swapping the IL (intracellular loop) of α4 with α1, when expressed with β2 and δ, produced receptors (α[414]β2δ) which were not inhibited by 3α,5β-THP when GABA-gated current was hyperpolarizing, while α[141]β2δ exhibited steroid-induced polarity-dependent modulation. Conclusions-These findings suggest that numerous neurosteroids exhibit polarity-dependent effects at α4β2δ GABARs which are dependent upon protein kinase C and the IL of α4.
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.
Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites
Nature, 2006
Inhibitory neurotransmission mediated by GABA A receptors can be modulated by the endogenous neurosteroids, allopregnanolone and tetrahydro-deoxycorticosterone 1 . Neurosteroids are synthesized de novo in the brain during stress 2 , pregnancy 3 and after ethanol consumption 4 , and disrupted steroid regulation of GABAergic transmission is strongly implicated in several debilitating conditions such as panic disorder, major depression, schizophrenia, alcohol dependence and catamenial epilepsy 3,5-8 . Determining how neurosteroids interact with the GABA A receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Here we identify two discrete binding sites in the receptor's transmembrane domains that mediate the potentiating and direct activation effects of neurosteroids. They potentiate GABA responses from a cavity formed by the a-subunit transmembrane domains, whereas direct receptor activation is initiated by interfacial residues between a and b subunits and is enhanced by steroid binding to the potentiation site. Thus, significant receptor activation by neurosteroids relies on occupancy of both the activation and potentiation sites. These sites are highly conserved throughout the GABA A receptor family, and their identification provides a unique opportunity for the development of new therapeutic, neurosteroid-based ligands and transgenic disease models of neurosteroid dysfunction.