We have reported that a novel GABA-A receptor (GABAR) isoform, alpha4-beta2-delta, is responsive to low, but not high, concentrations of ethanol. This effect was seen both in recombinant and native receptors following withdrawal from the GABA-modulatory steroid THP, when levels of this normally underexpressed receptor are markedly increased in limbic areas such as hippocampus. This proposal will investigate possible mechanisms for this effect, using transient expression in HEK-293 cells to examine dose-dependent effects of ethanol on single channel properties and deactivation kinetics. Because ethanol increases current amplitude at saturating concentrations of agonist, it is our hypothesis that ethanol is acting to increase gating efficacy of alpha4-beta-delta GABAR,as has been shown for steroid effects at this receptor. Therefore, site directed mutagenesis will be used to create constitutively active channels in GABAR expressed in HEK-293 cells to distinguish between effects on binding and gating. In addition, serine to tryptophan mutations at residue 270 in TM2 and A to S mutations in TM3 will be used to test whether residues in the binding cavity identified for effects of high dose ethanol acts to mediate the effects of low dose ethanol on alpha4-beta-delta GABAR. Other labs have reported effects of higher concentrations of ethanol which are effective at delta-containing GABAR. Therefore, it is also our hypothesis that the prolonged (2-5 min) pre-exposure to 1-3 mM ethanol can accelerate desensitization rate in the presence of 30 mM ethanol. Because alpha4-beta-delta GABAR are exclusively extrasynaptic, effects of low dose ethanol will be tested on tonic current recorded from dentate gyrus granule cells, which normally express high levels of these receptors. Antisense knock-down of alpha-4 and delta subunits to verify the role of alpha4-beta-delta GABAR in mediating effects of ethanol. The results from these studies may provide insight into potential mechanisms for effects of low dose ethanol at GABAR.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA012958-09
Application #
7741747
Study Section
Neurotoxicology and Alcohol Study Section (NAL)
Program Officer
Grandison, Lindsey
Project Start
2000-05-01
Project End
2011-11-30
Budget Start
2009-12-01
Budget End
2011-11-30
Support Year
9
Fiscal Year
2010
Total Cost
$269,930
Indirect Cost
Name
Suny Downstate Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
040796328
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Kuver, Aarti; Smith, Sheryl S (2016) Flumazenil decreases surface expression of ?4?2? GABAA receptors by increasing the rate of receptor internalization. Brain Res Bull 120:131-43
Sabaliauskas, Nicole; Shen, Hui; Molla, Jonela et al. (2015) Neurosteroid effects at ?4?? GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse. Brain Res 1621:170-86
Gong, Qi Hua; Smith, Sheryl S (2014) Characterization of neurosteroid effects on hyperpolarizing current at ?4?2? GABAA receptors. Psychopharmacology (Berl) 231:3525-35
Smith, Sheryl S (2013) ?4?? GABAA receptors and tonic inhibitory current during adolescence: effects on mood and synaptic plasticity. Front Neural Circuits 7:135
Smith, S S (2013) The influence of stress at puberty on mood and learning: role of the ?4?? GABAA receptor. Neuroscience 249:192-213
Shen, H; Mohammad, A; Ramroop, J et al. (2013) A stress steroid triggers anxiety via increased expression of ?4?? GABAA receptors in methamphetamine dependence. Neuroscience 254:452-75
Kuver, Aarti; Shen, Hui; Smith, Sheryl S (2012) Regulation of the surface expression of ?4?2? GABAA receptors by high efficacy states. Brain Res 1463:1-20
Sabaliauskas, Nicole; Shen, Hui; Homanics, Gregg E et al. (2012) Knockout of the ?-aminobutyric acid receptor subunit ?4 reduces functional ?-containing extrasynaptic receptors in hippocampal pyramidal cells at the onset of puberty. Brain Res 1450:11-23
Shen, Hui; Sabaliauskas, Nicole; Sherpa, Ang et al. (2010) A critical role for alpha4betadelta GABAA receptors in shaping learning deficits at puberty in mice. Science 327:1515-8
Zhou, Xiangping; Smith, Sheryl S (2009) Expression levels of the alpha4 subunit of the GABA(A) receptor in differentiated neuroblastoma cells are correlated with GABA-gated current. Neuropharmacology 56:1041-53

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