title: Characterization of neurosteroid effects on hyperpolarizing current at α4β2δ GABAA receptors.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.affiliation: Department of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY, 10023, USA.
authors.forename: Qi Hua
authors.initials: QH
authors.lastname: Gong
authors.forename: Sheryl S
authors.initials: SS
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D016764:Cell Polarity / Q000187:drug effects; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D018755:GABA Agonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000737:chemistry; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011280:Pregnanolone / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D018755:GABA Agonists; D007555:Isoxazoles; D050506:Mutant Chimeric Proteins; D018377:Neurotransmitter Agents; D011963:Receptors, GABA-A; D011280:Pregnanolone; C015542:gaboxadol
references.citation: Science. 2010 Mar 19;327(5972):1515-8
references.pmid: 20299596
references.citation: Front Neural Circuits. 2013 Sep 03;7:135
references.pmid: 24027497
references.citation: J Neurosci. 2002 May 15;22(10):RC223
references.pmid: 12006605
references.citation: J Neurosci. 2008 Nov 12;28(46):11890-9
references.pmid: 19005054
references.citation: J Neurosci. 2011 Jan 12;31(2):753-63
references.pmid: 21228184
references.citation: Curr Neuropharmacol. 2010 Mar;8(1):18-25
references.pmid: 20808543
references.citation: Nature. 2006 Nov 23;444(7118):486-9
references.pmid: 17108970
references.citation: Brain Res. 2012 Jun 29;1463:1-20
references.pmid: 22609410
references.citation: Biol Psychiatry. 2001 May 1;49(9):788-97
references.pmid: 11331087
references.citation: Nature. 2006 Apr 27;440(7088):1213-6
references.pmid: 16554753
references.citation: Nat Neurosci. 2007 Apr;10(4):469-77
references.pmid: 17351635
references.citation: Neuropharmacology. 2002 Sep;43(4):492-502
references.pmid: 12367596
references.citation: Biochem Biophys Res Commun. 1984 Apr 30;120(2):692-9
references.pmid: 6329179
references.citation: Biochem J. 1995 Apr 1;307 ( Pt 1):281-5
references.pmid: 7536413
references.citation: Br J Pharmacol. 2002 Aug;136(7):965-74
references.pmid: 12145096
references.citation: Neuropharmacology. 2009 Jan;56(1):141-8
references.pmid: 18760291
references.citation: J Neurosci. 2011 Dec 14;31(50):18198-210
references.pmid: 22171026
references.citation: Biol Pharm Bull. 2008 Sep;31(9):1646-50
references.pmid: 18758053
references.citation: Science. 1986 May 23;232(4753):1004-7
references.pmid: 2422758
references.citation: J Neurosci. 1996 Sep 1;16(17):5415-24
references.pmid: 8757254
references.citation: J Neurosci. 1982 Dec;2(12):1812-23
references.pmid: 6292381
references.citation: J Biol Chem. 2005 Sep 2;280(35):30741-50
references.pmid: 15970592
references.citation: J Biol Chem. 2000 Nov 17;275(46):36465-71
references.pmid: 10962006
references.citation: Neuroendocrinology. 1996 Feb;63(2):166-72
references.pmid: 9053781
references.citation: Nature. 1998 Apr 30;392(6679):926-30
references.pmid: 9582073
references.citation: J Neurosci. 2009 Aug 19;29(33):10416-23
references.pmid: 19692617
references.citation: J Neurophysiol. 1992 Jul;68(1):197-212
references.pmid: 1381418
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14602-7
references.pmid: 16984997
references.citation: Proc Natl Acad Sci U S A. 1991 May 15;88(10):4553-7
references.pmid: 1852011
references.citation: Alcohol Clin Exp Res. 2010 Mar 1;34(3):432-42
references.pmid: 20028362
references.citation: Front Neuroendocrinol. 2000 Jan;21(1):1-56
references.pmid: 10662535
references.citation: Psychoneuroendocrinology. 2004 Nov;29(10):1341-4
references.pmid: 15288714
references.citation: J Clin Endocrinol Metab. 2006 Aug;91(8):3092-9
references.pmid: 16720657
references.citation: J Neurosci. 1997 Jun 1;17(11):4022-31
references.pmid: 9151718
references.citation: J Physiol. 2012 Nov 15;590(22):5739-47
references.pmid: 22988137
references.citation: Nat Commun. 2011 Jul 05;2:376
references.pmid: 21730957
references.citation: Psychoneuroendocrinology. 2012 Apr;37(4):543-53
references.pmid: 21890277
references.citation: Brain Res. 1997 Aug 22;766(1-2):276-80
references.pmid: 9359616
references.citation: Br J Pharmacol. 2008 Mar;153(5):1062-71
references.pmid: 18204487
references.citation: J Neurosci. 2002 Mar 1;22(5):1541-9
references.pmid: 11880484
references.citation: J Biol Chem. 2010 Dec 31;285(53):41795-805
references.pmid: 20940303
references.citation: J Neurosci. 2003 Nov 19;23(33):10650-61
references.pmid: 14627650
references.citation: Neuroscience. 2013 Dec 19;254:452-75
references.pmid: 23994152
references.citation: J Neurophysiol. 2011 Oct;106(4):2057-64
references.pmid: 21795619
references.citation: J Neurosci. 2003 Nov 26;23(34):10934-43
references.pmid: 14645489
references.citation: J Clin Endocrinol Metab. 2000 Jul;85(7):2429-33
references.pmid: 10902789
references.citation: Mol Pharmacol. 1996 Jul;50(1):119-27
references.pmid: 8700104
references.citation: Nat Neurosci. 2007 Jan;10(1):40-8
references.pmid: 17159992
references.citation: J Endocrinol Invest. 1995 Dec;18(11):853-6
references.pmid: 8778157
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14439-44
references.pmid: 14623958
references.citation: Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13284-9
references.pmid: 9789080
references.citation: J Neurosci. 2008 Jan 2;28(1):106-15
references.pmid: 18171928
references.citation: Neuropharmacology. 2002 Sep;43(4):651-61
references.pmid: 12367610
references.citation: J Physiol. 2005 Apr 15;564(Pt 2):421-36
references.pmid: 15705652
references.citation: J Neurochem. 2002 Jul;82(1):110-7
references.pmid: 12091471
references.citation: J Neurosci. 1992 Mar;12(3):1040-62
references.pmid: 1312131