2004 — 2007 |
Eskandari, Sepehr |
S06Activity Code Description: To strengthen the biomedical research and research training capability of ethnic minority institutions, and thus establish a more favorable milieu for increasing the involvement of minority faculty and students in biomedical research. |
Molecular Physiology of Gamma-Aminobutyric Acid Transporters @ California State Poly U Pomona
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 use the Na + electrochemical gradient to transport GABA into cells after its release from nerve terminals and, thus, they regulate the concentration and lifetime of GABA in synaptic and extra-synaptic regions in the nervous system. In addition, they prevent spillover of GABA to surrounding synapses and, therefore, they ensure synaptic specificity. 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. Unfortunately, no drug exists which specifically targets GAT2 or GAT3, but we have recently identified a GAT4-specific inhibitor. We will express the GABA transporters in Xenopus laevis oocytes in order to address the following Specific Aims: (1) To use rapid concentration jumps at the GABA transporter binding pocket in order to gain mechanistic insight about ion and substrate binding and translocation across the plasma membrane. These experiments will use a novel rapid perfusion system developed in this laboratory, and will perform Na +, CI-, and GABA jumps at the transporter to gain a detailed understating of ligand interaction with the transporter. (2) To fully examine a novel Cl- channel mode identified in GAT4. These experiments will illuminate the significance of a novel Cl- channel mode, which we have recently identified in GAT4. (3) To fully characterize the action of a recently identified inhibitor with selectivity for GAT4. We have succeeded in identifying a novel specific inhibitor for GAT4. The experiments of this aim will fully characterize the inhibitory action of this agent, and will pave the way for determining the contribution of GAT4 to GABAergic inhibitory neurotransmission. (4) To formulate a substrate pharmacophore for the GABA transporters GAT3 and GAT4. As most studies have focused on GAT1, very little is known about the substrate binding pocket of GAT3 and GAT4. The experiments of this aim will identify lead compounds for future structure-guided design of specific inhibitors of GAT3 and GAT4.
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0.981 |
2009 — 2012 |
Eskandari, Sepehr |
SC1Activity Code Description: Individual investigator-initiated research projects aimed at developing researchers at minority-serving institutions (MSIs) to a stage where they can transition successfully to other s extramural support (R01 or equivalent). |
Molecular Physiology of Y-Aminobutyric Acid Transporters @ California State Poly U Pomona
DESCRIPTION (provided by applicant): 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.
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0.981 |