Qi Hua Gong1, Sheryl S Smith. 1. Department of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY, 10023, USA.
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.
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.
Authors: Hal A Droogleever Fortuyn; Frank van Broekhoven; Paul N Span; Torbjörn Bäckström; Frans G Zitman; Robbert J Verkes Journal: Psychoneuroendocrinology Date: 2004-11 Impact factor: 4.905