Date of Graduation
5-2015
Document Type
Dissertation (PhD)
Program Affiliation
Biochemistry and Molecular Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Vasanthi Jayaraman, Ph.D.
Committee Member
Jianping Jin, Ph.D.
Committee Member
John O'Brien, Ph.D.
Committee Member
John A. Putkey, Ph.D.
Committee Member
John L. Spudich, Ph.D.
Abstract
N-methyl-D-aspartate (NMDA) receptors are one of the three main types of ionotropic glutamate receptors in the central nervous system. NMDA receptors mediate the rapid excitatory neurotransmission that underlies learning and memory formation. Conversely, NMDA receptors are implicated in a variety of neurological disorders. Studies targeting the mechanism of allosteric modulation, such as this study, hope to contribute to the understanding of how NMDA receptors are modulated to allow for better drug development.
NMDA receptors are obligate heterotetramers, typically composed of glycine-binding GluN1 subunits and glutamate-binding GluN2 subunits. The GluN2 subunits can be one of four subtypes (A-D). Each subunit is organized into distinct domains: extracellular amino-terminal (ATD) and ligand-binding domains, the transmembrane, pore-forming domain, and an intracellular carboxyl-terminal domain. The ATD includes the binding site of a number of allosteric modulators. Endogenous modulators such as the zinc cation or spermine can inhibit and potentiate current through the receptor, respectively. The synthetic compound ifenprodil also binds the ATDs and inhibits the receptor. We used luminescence resonance energy transfer to study the structural changes accompanying modulator binding and then linked those conformational changes to the function of the receptor using electrophysiology. LRET measurements were made between subunits to determine the architecture of the ATDs in an assembled receptor.
Our studies reveal that the ATDs have a compact overall organization, and that the inherent conformation of a GluN1 or GluN2 ATD varies in a fashion that correlates with the open channel probability of the receptor. Additionally, modulator binding to the ATDs induces conformational changes. Inhibitors, such as zinc and ifenprodil, induce a closure in the GluN2 ATD, whereas the potentiator spermine induces an opening of the GluN2 ATD. The lower lobes of the ATDs undergo a rotational movement when any allosteric modulator was bound, suggesting the rotation of the domain is independent of the open/close mechanism. These studies reveal that the receptor can be modulated by influencing the conformation of the ATD.
Keywords
NMDA Receptor, Allosteric Modulation, Zinc, Ifenprodil, Spermine, Structural Mechanism, Luminescence Resonance Energy Transfer, Structure-Function