Gamma-aminobutyric acid (GABA) is released from presynpatic terminals and binds to postsynaptic GABA receptors. The binding of GABA on the extracellular amino terminal domain leads to a structural change at the pore, buried deep within the membrane. This structural change at the pore permits the flux of chloride down its concentration gradient, thereby inhibiting the postsynaptic neuron. The general aim of this project is to understand the mechanism by which this binding of GABA leads to the gating of the pore. For these studies we use recombinant receptors expressed in oocytes and, where necessary, other exogenous expression systems. We have assembled a variety of techniques that will allow us to probe the activation mechanism from both a structural and functional vantage point.
The first aim will use mutagenesis, cysteine accessibility, and electrophysiological techniques to identify the key domains that play a role in activation. This will set the stage for the next two aims that will investigate the dynamics of the activation process. For example, the second aim will use site-specific fluorescence labeling in combination with two-electrode voltage clamp to identify functionally-relevant molecular rearrangements.
The third aim employs a novel approach in which charged amino acids are substituted at select positions to identify parts of the receptor within the membrane field that move during activation. GABA is the major inhibitory neurotransmitter in the mammalian brain. Dysfunctions in GABA-mediated inhibition have been implicated in the etiology of a variety of brain disorders. Furthermore, many clinicallyprescribed compounds (barbiturates, benzodiazepines, neurosteroids) exert their therapeutic effects by modulating the function of GABA receptors. Crucial to understanding the molecular basis of these diseases and drugs is a detailed understanding of the molecular mechanism of activation. The results from this proposal should facilitate this endeavor.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37NS035291-09
Application #
6686939
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Stewart, Randall
Project Start
1995-08-01
Project End
2007-06-30
Budget Start
2003-07-15
Budget End
2004-06-30
Support Year
9
Fiscal Year
2003
Total Cost
$344,375
Indirect Cost
Name
University of Alabama Birmingham
Department
Neurosciences
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Li, Ping; Khatri, Alpa; Bracamontes, John et al. (2010) Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids. Mol Pharmacol 77:539-46
Zheleznova, Nadezhda N; Sedelnikova, Anna; Weiss, David S (2009) Function and modulation of delta-containing GABA(A) receptors. Psychoneuroendocrinology 34 Suppl 1:S67-73
Zheleznova, N; Sedelnikova, A; Weiss, D S (2008) alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids. Br J Pharmacol 153:1062-71
Wotring, Virginia E; Weiss, David S (2008) Charge scan reveals an extended region at the intracellular end of the GABA receptor pore that can influence ion selectivity. J Gen Physiol 131:87-97
Sedelnikova, Anna; Smith, Craig D; Zakharkin, Stanislav O et al. (2005) Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model. J Biol Chem 280:1535-42
Erkkila, Brian E; Weiss, David S; Wotring, Virginia E (2004) Picrotoxin-mediated antagonism of alpha3beta4 and alpha7 acetylcholine receptors. Neuroreport 15:1969-73
Wotring, V E; Miller, T S; Weiss, D S (2003) Mutations at the GABA receptor selectivity filter: a possible role for effective charges. J Physiol 548:527-40
Torres, Viviana I; Weiss, David S (2002) Identification of a tyrosine in the agonist binding site of the homomeric rho1 gamma-aminobutyric acid (GABA) receptor that, when mutated, produces spontaneous opening. J Biol Chem 277:43741-8
Chang, Yongchang; Ghansah, Emmanuel; Chen, Yonghui et al. (2002) Desensitization mechanism of GABA receptors revealed by single oocyte binding and receptor function. J Neurosci 22:7982-90
Ratra, Gurpreet S; Erkkila, Brian E; Weiss, David S et al. (2002) Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor. Toxicol Lett 129:47-53

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