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Date of Award

Spring 2013

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. Lisa Gentile

Abstract

The ionotropic glutamate receptor family is composed of four unique classes of transmembrane channels: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D- aspartate (NMDA), kainate and delta. This family of ligand-gated ion channels requires the binding of glutamate (in addition to glycine for NMDA receptors) to allow an influx of cations into the post-synaptic neuron. Like any cellular process, the excitatory pathway of ionotropic glutamate receptors is highly regulated through internal mechanisms. The endogenous neurosteroids pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS) are nearly identical structurally, yet differentially regulate certain ionotropic glutamate receptors allosterically upon their binding. Electrophysiology research has shown that while PS has a potentiating effect on NMDA receptors containing GluN2A or GluN2B subunits, the same neurosteroid has an inhibitory effect on non-NMDA receptors and NMDA receptors instead containing GluN2C or GluN2D subunits. Furthermore, PREGAS inhibits the entire ionotropic glutamate receptor family regardless of subunit composition.

In this paper, results are presented that, when combined with prior studies in our lab, aim to elucidate the dissimilar regulatory properties of the neurosteroids PS and PREGAS on NMDA receptors. The research described here is focused on the extracellular amino terminal domain (ATD) of GluN2B, one of four NMDA GluN2 subunits. Intrinsic fluorescence spectroscopy shows that both neurosteroids bind to the GluN2B ATD. In addition, PS and PREGAS have similar binding affinities for this subunit domain, as determined from their respective dissociation constants (KD). Site-directed mutagenesis suggests that PS and PREGAS bind to different sites on the GluN2B ATD, with PS conceivably binding in the known ifenprodil binding site. The binding of PS and PREGAS on the GluN2B ATD is characterized here in an effort to provide results that will add to the scholarship of compounds being designed and investigated for usage in treating mental illness and neurological disease states.

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