Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant alpha1beta2gamma2 GABAA receptors.
Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. 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 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.
Department of Anesthesiology, Washington University in St. Louis, Campus Box 8054, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Ping
P
Li
Douglas F
DF
Covey
Joe Henry
JH
Steinbach
Gustav
G
Akk
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D018757:GABA Modulators / Q000737:chemistry / Q000378:metabolism*; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D006801:Humans; D009154:Mutation; D018408:Patch-Clamp Techniques; D011083:Polycyclic Compounds / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects*; D013256:Steroids / Q000737:chemistry / Q000494:pharmacology
D018757:GABA Modulators; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; C546240:GABRA1 protein, human; C546279:GABRG2 protein, human; C546242:Gabra1 protein, rat; C502185:Gabrb2 protein, rat; C546281:Gabrg2 protein, rat; D011083:Polycyclic Compounds; D011963:Receptors, GABA-A; D013256:Steroids; C511625:dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz(e)indene-3-carbonitrile