Year of Award

2007

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Pharmaceutical Sciences

Other Degree Name/Area of Focus

Pharmacology

Department or School/College

College of Health Professions and Biomedical Sciences

Committee Chair

Richard J. Bridges

Commitee Members

Michael P. Kavanaugh, John M. Gerdes, Charles M. Thompson, Keith Parker, David Poulsen

Keywords

computational modeling, glutamate, neurosteroid, uptake

Abstract

L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS that can mechanistically contribute to either neuronal signaling or neuronal pathology. Consequently, its concentration in the CNS must be carefully regulated, a critical need that is met by the excitatory amino acid transporters (EAATs). The presence of at least five isoforms of EAATs raises interesting questions as to potential structural and functional differences among the subtypes. We have investigated possible differences in the ligand binding domains of the EAATs through the development of computationally based pharmacophore models. An EAAT2-specific model was created with four potent and selective ligands that act as non-substrate inhibitors: cis-5-methyl-L-trans-2,3-pyrrolidine dicarboxylate, L-anti-endo-3,4-methano-pyrrolidine-3,4-dicarboxylate (L-anti-endo-3,4-MPDC), (2S,3R,4S)-2-(carboxy-cyclopropyl) glycine (L-CCG-IV) and L-B-threo-benzyloxy-aspartate (L-B-TBOA). This model predicts distinct regions that might influence the potency and selectivity of EAAT2 ligands, including: 1) a highly conserved positioning of the two carboxylate and the amino groups, 2) a nearby region that can accommodate selective modifications (e.g., cyclopropyl ring, CH3 groups, and O atoms), and 3) a region occupied by the benzyl ring of L-B-TBOA. This model was also used in conjunction with L-B-threo-benzyl aspartate (L-B-TBA), a recently characterized preferential inhibitor of EAAT3, to identify possible differences between EAAT2 and EAAT3.

Functional studies on the EAATs also led to the identification of a putative modulatory mechanism that is specific for EAAT1. Thus, a series of sulfated neuroactive steroids, including pregnenolone sulfate (PREGS), were found to selectively increase the ability of EAAT1 to transport atypical substrates like D-aspartate and L-cysteine, but not L-glutamate. The effect was rapid, reversible, limited to a select group of sulfated steroids, and not observed with either EAAT2 or EAAT3. Interestingly, the action of PREGS could be blocked by the simultaneous addition of arachidonic acid, a previously recognized inhibitory modulator of EAAT1. The fact that this observed change in activity was produced by neurosteroids raises questions not only related to the regulatory mechanisms itself, but also to the possible role of neurosteroid in modulating glutamate transport.

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© Copyright 2007 Shailesh Ramjilal Agarwal