The plasmalemmal serotonin transporter (SERT) terminates serotonergic neurotransmission and thus plays a critical role in shaping the duration and magnitude of synaptic signaling. It works by coupling preexisting ion gradients to the thermodynamically unfavorable movement of serotonin from the synapse to neuronal and glial cytoplasms. This molecular machine has garnered significant clinical attention because its dysfunction has been implicated in multiple debilitating neuropsychiatric diseases such as depression, autism, obsessive- compulsive disorder, and generalized anxiety. Furthermore, it is the target of numerous psychoactive agents such as antidepressants, cocaine, and the amphetamine derivative "ecstasy". Despite such clinical and pharmacological significance, atomic-level detail into the mechanism of substrate translocation, inhibition, and regulation has remained elusive. Two goals of this proposal include 1) solving the structure of a SERT in complex with substrates, ions, and inhibitors;and 2) probing the conformational dynamics of these interactions via hydrogen-deuterium exchange mass spectrometry (HDX-MS). Structure-based hypotheses will subsequently be tested with a combination of site-directed mutagenesis, cysteine crosslinking, transient and steady-state flux kinetics, dissociation/association binding kinetics, X-ray crystallography, and HDX-MS. Success will permit further investigation into specific antagonist binding sites and perhaps into the molecular basis for drug resistance, thereby paving the way for rational drug design efforts. When eventually coupled with in vivo work beyond the scope of this proposal, SERT structure/function studies may also shed light on the molecular underpinnings of disease-associated polymorphisms. Achieving any of one of these objectives would likely have significant impact in both the laboratory and the clinic.

Public Health Relevance

The serotonin transporter (SERT) plays a critical role in terminating serotonergic neurotransmission and thus in shaping the duration and magnitude of synaptic signaling. Its dysfunction has been implicated in multiple neuropsychiatric diseases and it is the target of numerous psychoactive compounds. Thus, elucidating transport mechanism, inhibition, and pharmacology are crucial goals, all objectives of this grant application.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
1R01MH100688-01A1
Application #
8773997
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Nadler, Laurie S
Project Start
2014-08-15
Project End
2019-05-31
Budget Start
2014-08-15
Budget End
2015-05-31
Support Year
1
Fiscal Year
2014
Total Cost
$578,660
Indirect Cost
$228,870
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520