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Dual Potentiating and Inhibitory Actions of a Benz[e]indene Neurosteroid Analog on Recombinant 122 GABA_A Receptors

Departments of Anesthesiology (P.L., J.H.S., G.A.) and Molecular Biology and Pharmacology (D.F.C.), Washington University in St. Louis, St. Louis, Missouri

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-(3,3a,5a,7,9a,9b)]-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 1, 2, and 2L 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 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the 1 subunit Q242W and the double mutation 1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 µM), 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 µM^-1 s^-1. Based on the kinetic mechanism of action, and the finding that this effect is blocked by the 1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.

Address correspondence to: Dr. Gustav Akk, Department of Anesthesiology, Washington University in St. Louis, Campus Box 8054, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: akk{at}morpheus.wustl.edu

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