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Characterization of neurosteroid effects on hyperpolarizing current at α4β2δ GABAA receptors

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Abstract

Rationale

The neurosteroid 3α,5β-THP (3α-OH-5β-pregnan-20-one, pregnanolone) is a modulator of the GABAA 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.

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Acknowledgments

This study was supported by grants from the U.S. National Institutes of Health: DA09618, AA12958 and MH100561 to S.S.S. We also thank D. H. Smith, M. Yuan, M. Dattilo and A. Kuver for helpful technical assistance.

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  1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY, 10023, USA

    Qi Hua Gong & Sheryl S. Smith

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  1. Qi Hua Gong
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  2. Sheryl S. Smith
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Correspondence to Sheryl S. Smith.

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Gong, Q.H., Smith, S.S. Characterization of neurosteroid effects on hyperpolarizing current at α4β2δ GABAA receptors. Psychopharmacology 231, 3525–3535 (2014). https://doi.org/10.1007/s00213-014-3538-x

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  • DOI: https://doi.org/10.1007/s00213-014-3538-x

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