Author ORCID Identifier

0000-0002-2438-362X

Date of Graduation

8-2017

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Vasanthi Jayaraman

Committee Member

Darren Boehning

Committee Member

Jeffrey Frost

Committee Member

Shane Cunha

Committee Member

John Spudich

Abstract

Structural and Functional Effect of Phosphorylation on AMPA Receptors

Cailtin Edmunds show you, BA

Advisory Professor: Vasanthi Jayaraman, Ph. D.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is the primary contributor to neuronal fast excitatory transmission, and plays a key role in learning and memory. Previous studies have established that residues S818, S831, and T840 in the C-terminal segment of GluA1 homomeric AMPA receptor are phosphorylated by PKC, and phosphorylation at these sites leads to an increase in receptor conductance. We show that the domain inclusive of those sites alters its secondary structure due to phosphorylation (using glutamate substitution as a mimic) in a lipid charge dependent manner using Fourier transform infrared spectroscopy. We also indicate a strong shift in the domain’s conformational landscape using single molecule FRET. Using wild-type GluA1 receptors, as well as glutamate and alanine to mimic phosphorylation and dephosphorylation respectively, we show based on FRET measurements that the C-terminal segment moves away from the membrane upon phosphorylation. Additionally, the FRET between the C-terminal segment and inner leaflet of the plasma membrane increases upon activation of the receptor for the triple alanine dephosphorylated receptor indicating a further motion towards the membrane associated with activation of the receptor. The phosphomimetic receptor, on the other hand, shows no change in FRET associated with activation of the receptor. However, addition of the lipid sphingosine restores this change in FRET due to activation for the phosphomimetic receptor. Using single channel current recordings we confirm that there is an increase in all conductance levels of the AMPA receptor in the phosphomimetic receptor and show that this increase in conductance is reversed by incorporation of sphingosine in the membrane or addition of poly-L-lysine. Thus using FRET and functional measurements we show that the plasma membrane lipid content is critical in mediating the phosphorylation mediated functional changes in GluA1 AMPA receptor.

Keywords

AMPA Receptor, GluA1, GluR1, Phosphorylation, FTIR, Microscopy, Vibrational Spectroscopy, single molecule FRET, single cell imaging, auxiliary subunits, lipids, vesicles, peptides

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