Our long-term objective is to gain a comprehensive understanding of the molecular mechanism by which gamma-aminobutyric acid (GABA) transporters accomplish Na+/Cl-/GABA cotransport across the plasma membrane. The GABA transporters transport GABA into cells after its release from nerve terminals and, thus, they regulate the concentration profile and lifetime of GABA in synaptic and extra-synaptic regions of the nervous system. GABA is the most abundant inhibitory neurotransmitter in the central nervous system and, therefore, potentiation of GABAergic neurotransmission via inhibition or reversal of the GABA transporters is believed to have therapeutic value in treating epileptic seizures and stroke. Four GABA transporter isoforms are present in the mammalian brain (GAT1, GAT2, GAT3, and GAT4), and exhibit significant differences in function, pharmacology, and localization. Indeed, the GABA transporters have been implicated in epilepsy, and one isoform (GAT1) is the target of the anti-epileptic drug tiagabine. Our understanding of the function and pharmacology of GAT2, GAT3, and GAT4 lags far behind that of GAT1. The GABA transporters belong to the neurotransmitter:Na+ symporter family, and a recent high-resolution crystal structure of a bacterial family member (leucine transporter, LeuTAa) has dramatically enhanced our understanding of structure and function of these transporters. The crystal structure has also paved the way for detailed structure-function characterization of the GABA transporters. We will express the GABA transporters in Xenopus laevis oocytes in order to address the following Specific Aims: (1) To combine electrophysiological and electron microscopic measurements in order to determine the physiological turnover rate of GAT2, GAT3, and GAT4. (2) To use site-directed mutagenesis in order to identify functional sites that distinguish the kinetic phenotypes of GABA transporter isoforms GAT3 and GAT4. (3) To use molecular modeling techniques in conjunction with experimental screening assays in order to gain mechanistic insight regarding substrate coordination in the binding site of the GABA transporters GAT3 and GAT4. Gamma-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the brain. The brain extracellular concentration of GABA is regulated by four GABA transporter isoforms, only one of which is the target of the antiepileptic drug tiagabine. The proposed research examines the mechanism of function of GABA transporters and will pave the way for the development of novel isoform-specific antiepileptic drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Enhancement Award (SC1)
Project #
5SC1GM086344-04
Application #
8209038
Study Section
Special Emphasis Panel (ZGM1-MBRS-0 (NP))
Program Officer
Dunsmore, Sarah
Project Start
2009-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$316,305
Indirect Cost
$93,555
Name
California State Polytechnic University Pomona
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
028929438
City
Pomona
State
CA
Country
United States
Zip Code
91768
Paroder-Belenitsky, Monika; Maestas, Matthew J; Dohan, Orsolya et al. (2011) Mechanism of anion selectivity and stoichiometry of the Na+/I- symporter (NIS). Proc Natl Acad Sci U S A 108:17933-8
Anderson, Cynthia M; Kidd, Patrick D; Eskandari, Sepehr (2010) GATMD: ýý-aminobutyric acid transporter mutagenesis database. Database (Oxford) 2010:baq028
Matthews Jr, Edward; Rahnama-Vaghef, Ali; Eskandari, Sepehr (2009) Inhibitors of the gamma-aminobutyric acid transporter 1 (GAT1) do not reveal a channel mode of conduction. Neurochem Int 55:732-40
Eskandari, Sepehr (2009) Remarkable commonalities of electrogenic and electroneutral Na+-phosphate cotransporters. J Physiol 587:4131-2