Date of Award

1-1-2017

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Content Description

1 online resource (ii, viii, 54 pages)

Dissertation/Thesis Chair

Li Niu

Committee Members

Jia Sheng, Ting Wang, Alexander Shekhtman

Keywords

AMPA receptors, CX546, GluA2Qflip, Laser-Pulse Photolysis, Potentiators, Whole-cell Current Recording, Neurotransmitter receptors, Ion channels, Neural transmission, Laser photochemistry, Glutamic acid

Subject Categories

Biochemistry | Chemistry | Neuroscience and Neurobiology

Abstract

Insufficient activity of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate ion channels is involved in neurological disorders and developmental problems, such as schizophrenia, one of the most serious psychiatric diseases. Use of regulatory agents to enhance AMPA receptor activity under the circumstance has been shown therapeutically beneficial. Specifically, small-molecule compounds generally termed as potentiators or positive modulators of AMPA receptors have long been pursued as drug candidates for a potential treatment of these neurological disorders and diseases. The goal of my MS thesis work is to investigate the mechanism of potentiation on AMPA receptor by a classic potentiator known as CX546. The hypothesis to be tested is that CX546, as an example, affects the channel-opening kinetics as a way to increase whole-cell current amplitude – such a measure is a way to link the amount of ions passing through the channel when the channel is opened by the binding of glutamate. In my study, I expressed AMPA receptors, one at a time, in human embryonic kidney cells (HEK-293 cells). Using whole-cell recording, I characterized the AMPA receptor activity in the absence and presence of CX546. My results show that CX546 augmented the amplitude of the whole-cell current response to glutamate, the endogenous neurotransmitter of AMPA receptors, only through the closed-channel, not the open-channel, conformation of the AMPA receptors. In selectivity assay, my study shows that CX546 does not exhibit appreciable action (i.e., either potentiation or inhibition) on either kainate or NMDA receptors, the two other subtypes of the glutamate ion channel family. Furthermore, using a laser-pulse photolysis technique and “caged” glutamate (i.e., a photolabile precursor of glutamate), I also characterized the effect of CX546 on the channel-closing rate constant. I observed that CX546 slows the rate of channel closing as compared with the control; the control is the GluA2Qflip homomeric AMPA receptor channels. Given all the data I have obtained, the most plausible mechanism of potentiation can be explained by the following mechanism: the decrease of channel-closing rate allows a larger amount of ion passage through the open channel, thereby causing a greater whole-cell current response at the same glutamate concentration. The new mechanistic revelation obtained from my study provides useful implications in rational design of mechanism-based, more potent and more selective potentiators for AMPA receptors.

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