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Title page for ETD etd-03252019-134248


Type of Document Dissertation
Author Quinlan , Meagan Anne
Author's Email Address meagan.quinlan@gmail.com
URN etd-03252019-134248
Title Structural and Functional Dynamics of Serotonin Transporter Gene Variants
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Brian Wadzinski, PhD Committee Chair
Ana Carnerio, PhD Committee Member
Heidi Hamm, PhD Committee Member
Kevin Schey Committee Member
Randy Blakely, PhD Committee Member
Keywords
  • Serotonin
  • Autism Spectrum Disorder
  • Serotonin Transporter
Date of Defense 2019-03-14
Availability restrictone
Abstract
The antidepressant-sensitive serotonin (5-HT) transporter (Slc6a4; SERT) is a dynamically regulated membrane protein that tightly controls the signaling potential of 5-HT by rapidly clearing 5-HT from the extracellular space. Mechanistically, SERT translocate 5-HT across the membrane via an alternating-access mechanism, whereby 5-HT binds to the transporter when the 5-HT binding site is exposed to the extracellular space (outward-facing), leading to a conformational shift of the transporter to close gates that prevent dissociation of 5-HT and opening of gates that allow access of the 5-HT binding site to the cytosolic space (inward-facing). This process repeats after the transporter reorients to expose an unloaded 5-HT binding site to the extracellular. Previously, we found activation of the innate immune system, through a p38⍺ MAPK signaling pathway, shifts the transporter’s externally exposed 5-HT binding site to a high-affinity state (SERT*) that leads to an enhanced 5-HT rate of clearance. It appears that the SERT* state is constitutively-induced by two autism spectrum disorder (ASD)-associated SERT coding variants, SERT Ala56 and SERT Asn605, which are also insensitive to p38⍺ MAPK and protein kinase G (PKG) signaling. Here we show, using in vitro biochemical, as well as ex vivo physiological approaches, that the SERT Ala56 and SERT Asn605 variants stabilize an outward-facing conformation that can provide a mechanism for altered catalytic regulation of both variants, and constitutively increased 5-HT uptake observed with SERT Ala56. Proteomic analyses provide evidence that SERT Ala56 differentially interacts in vivo with proteins associated with ASD and proteins that affect SERT scaffolding and post-translational modifications (PTMs). To further explore the molecular features of SERT regulation in vivo, we used CRISPR/Cas9 approaches to generate the phosphorylation insensitive knock-in mouse, SERT Ala276, a residue implicated in PKG and p38⍺ MAPK regulation. Preliminary findings show SERT Ala276 mice demonstrate changes in social interactions and obsessive behavior. Overall, we hypothesize SERT conformation equilibrium can be shifted transiently in response to stimulation of signaling pathways or constitutively by engineered or disease-associated coding variants, which impact transporter PTMs and protein-protein interactions, ultimately affecting SERT activity.
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