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Effects of neuroactive steroids on the recombinant GABAA receptor in Xenopus oocyte
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction: Neuroactive steroids represent a class of both synthetic and naturally occurring steroids that have an effect on neural function. In addition to classical genomic mechanism by the hormones progesterone, deoxycorticosterone and testosterone 3α-OH metabolites of these hormones enhance GABAA receptor through rapid non-genomic mechanism. The site(s) of action of these neuroactive steroids namely 3α-OH-5α-pregnan-20 one, (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and 5α androstane-3α,17β-diol on GABAA receptor are distinct from that of benzodiazepines and barbiturate binding sites. The modulation site(s) has a well-defined structure activity relationship with a 3α-hydroxy and a 20-ketone configuration in the pregnane molecule required for agonistic action. Pregnenolone sulfate is a noncompetitive GABAA receptor antagonist and inhibit GABA activated Cl- current in an activation dependant manner. 3β-hydroxy A-ring reduced pregnane steroids are also GABAA receptor antagonist and inhibit GABAA receptor function and its potentiation induced by their 3α-diesteromers in a noncompetitive manner.

Aim: The aim was to investigate if the effect of GABA, pentobarbital antagonism by bicuculline and if the effect of GABA-agonist and antagonist neuroactive steroids including pregnenolone sulfate is dependant on the α-subunits of GABAA receptor. Furthermore, the studies aimed at investigating the binding site of pregnenolone sulfate and if its effect is dependent on γ-subunit. In addition, the inhibitory effect of pregnenolone sulfate and 3β-hydroxy steroids has been characterized. We also wanted to investigate if the neuroactive steroids effect vary between the human and rat recombinant α1β2γ2L receptors and between the long (L) and short (S) variants of γ2-subunit.

Method: Experiments were performed by the two electrodes voltage-clamp technique using oocytes of Xenopus laevis expressed with recombinant GABAA receptors containing α1, α4 or α5, β2, γ2L and γ2S-subunits.

Results: There was no difference between the α1, α4 and α5-containing subunits regarding GABA and pentobarbital inhibition by bicuculline. GABA-activated current in the binary αβ was potent than that of ternary αβγ receptor. Unlike Zn2+ effect, inhibition by pregnenolone sulfate on the GABAA receptor is not dependant on the γ-subunit. It is likely that the 2’ residue closest to the N-terminus of the protein at M2 helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl- channels. Point mutation at M2 helix of the β2-subunit (b2A252S) can dramatically reduce the inhibitory effect of PS on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. Agonist and antagonist steroids also varied in their efficacy between the human and rat α1β2γ2L receptor. Neuroactive steroids also showed difference between human γ2L and γ2S-containing receptor.

Conclusions: GABA and pentobarbital antagonism by bicuculline is not dependant on α-subunit. Pregnenolone sulfate binding site is different from that of Zn2+. 3β-hydroxysteroids and pregnenolone sulfate inhibit GABAA receptor through different mechanisms. Neuroactive steroids also differ between species and between the long and short variant of γ- subunit.

Place, publisher, year, edition, pages
Umeå: Medicinsk fakultet , 2007. , 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1099
Keyword [en]
GABA, GABAA receptor, neuroactive steroids
National Category
Clinical Science
Identifiers
URN: urn:nbn:se:umu:diva-1112ISBN: 978-91-7264-273-7 (print)OAI: oai:DiVA.org:umu-1112DiVA: diva2:140241
Public defence
2007-05-25, D, by 1D, Tr 9 (tandläkerhögskolan), NUS, 13:00 (English)
Opponent
Supervisors
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2016-06-15Bibliographically approved
List of papers
1. GABA-site antagonism and pentobarbital actions do not depend on the α-subunit type in the recombinant rat GABA-A receptor
Open this publication in new window or tab >>GABA-site antagonism and pentobarbital actions do not depend on the α-subunit type in the recombinant rat GABA-A receptor
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2006 (English)In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 187, no 4, 479-488 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2300 (URN)10.1111/j.1748-1716.2006.01593.x (DOI)
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2017-12-14Bibliographically approved
2. Pregnenolone sulfate and zinc inhibit recombinant rat GABAA receptor through different channel property
Open this publication in new window or tab >>Pregnenolone sulfate and zinc inhibit recombinant rat GABAA receptor through different channel property
2006 (English)In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 188, no 3/4, 153-163 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2301 (URN)
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2017-12-14Bibliographically approved
3. 3Beta-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABA(A) receptor through different channel property.
Open this publication in new window or tab >>3Beta-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABA(A) receptor through different channel property.
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2007 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 557, no 2-3, 124-131 p.Article in journal (Refereed) Published
Keyword
Animals, Chloride Channels/*metabolism, Dose-Response Relationship; Drug, Female, GABA Antagonists/*pharmacology, GABA Modulators/pharmacology, Hydroxysteroids/*pharmacology, Inhibitory Concentration 50, Kinetics, Oocytes/metabolism, Patch-Clamp Techniques/methods, Pregnenolone/*pharmacology, Rats, Receptors; GABA-A/*antagonists & inhibitors/genetics, Recombinant Proteins/pharmacology, Structure-Activity Relationship, Xenopus laevis
Identifiers
urn:nbn:se:umu:diva-16916 (URN)10.1016/j.ejphar.2006.11.071 (DOI)17239367 (PubMedID)
Available from: 2008-01-12 Created: 2008-01-12 Last updated: 2017-12-14Bibliographically approved
4. Functional difference between recombinant human and rat α1β2γ2L GABAA receptors
Open this publication in new window or tab >>Functional difference between recombinant human and rat α1β2γ2L GABAA receptors
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2303 (URN)
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2016-06-15Bibliographically approved

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