Neurotransmitter:sodium symporters (NSS) couple the accumulation of substrate to the movement of sodium ions down their concentration gradient across the plasma membrane, and as such constitute key elements in cellular signaling and homeostasis. NSS include the transporters for dopamine, serotonin and norepinephrine-targets for amphetamine, cocaine, and antidepressant drugs-as well as the transporters for GABA and glycine, which are targeted for treatment of epilepsy and schizophrenia. In 2005 the Gouaux group solved at 1.65 ? the structure of LeuT, a bacterial NSS homolog, crystallized with 1 Leu and 2 Na+ bound in an occluded binding pocket (referred to as primary substrate binding (S1) site). The structure provided no easy clues to the pathway of substrate to the S1 site from the extracellular or the intracellular side. An unexpected second substrate binding (S2) site located in the extracellular vestibule was identified during the previous project period;binding and flux experiments showed that the two binding sites can be occupied simultaneously. Substrate in the S2 site allosterically triggers intracellular release of Na+ and substrate from the S1 site, thereby functioning as a "symport effector." Because tricyclic antidepressants (TCA) bind differently to this S2 site, they do not promote substrate release from the S1 site and thus act as symport uncouplers to inhibit transport. Identifying the conformational changes associated with transport and the permeation pathways that are formed within the transporter are long term goals of this project critical to understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. To achieve this goal, an integrated approach has been developed based on active collaborations with investigators whose expertise in computational modeling (Harel Weinstein), membrane protein crystallography (Poul Nissen), and single-molecule fluorescence spectroscopy (Scott Blanchard) enables the combined multidisciplinary approach described in this application. The following specific aims are proposed: 1) To use our novel discoveries regarding the specificity and modulation of S2 binding, by detergents, mutations, and ionic substitution, to develop conditions that enable us to understand the regulation of LeuT properties by the S2 binding site and to solve a structure of LeuT with substrate bound to the S2 site. This will provide atomic resolution data to inform our mechanistic hypothesis as to the essential role in transport of substrate binding to this site. 2) To characterize the mechanism of substrate transport in terms of specific conformational changes in the transporter that propagate the allosteric signal triggered by substrate binding to the S2-site towards the intracellular gate of the transporter and allow inward release of substrate. 3) To establish the relevance of our structural and functional findings in bacterial transporters to understanding the function of SERT and DAT. We will: a) demonstrate the essential functional role of the S2 site in these human transporters, and b) use a Cl-- dependent LeuT mutant to determine the structure of the Cl- binding site and thus to explicate the functional role of Cl- in SERT and DAT.

Public Health Relevance

Neurotransmitter transporters are the target of psychostimulant drugs such as cocaine and amphetamine and are targets for antidepressants as well as for new drugs in development for the treatment of epilepsy and schizophrenia. Identifying the conformational dynamics of transport, the permeation pathways within the transporter, and the role of substrate and inhibitor binding are critical for understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. The powerful approaches we have established will allow us to reach this understanding and address drug action and guidelines for therapy design anchored in solid structural and functional information.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA017293-10
Application #
8477155
Study Section
Special Emphasis Panel (ZRG1-MDCN-J (02))
Program Officer
Hillery, Paul
Project Start
2003-09-30
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
10
Fiscal Year
2013
Total Cost
$605,286
Indirect Cost
$151,615
Name
Columbia University (N.Y.)
Department
Psychiatry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Malinauskaite, Lina; Quick, Matthias; Reinhard, Linda et al. (2014) A mechanism for intracellular release of Na+ by neurotransmitter/sodium symporters. Nat Struct Mol Biol 21:1006-12
LeVine, Michael V; Weinstein, Harel (2014) NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT. PLoS Comput Biol 10:e1003603
Dehnes, Yvette; Shan, Jufang; Beuming, Thijs et al. (2014) Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation. Neurochem Int 73:4-15
Mondal, Sayan; Khelashvili, George; Weinstein, Harel (2014) Not just an oil slick: how the energetics of protein-membrane interactions impacts the function and organization of transmembrane proteins. Biophys J 106:2305-16
Kantcheva, Adriana K; Quick, Matthias; Shi, Lei et al. (2013) Chloride binding site of neurotransmitter sodium symporters. Proc Natl Acad Sci U S A 110:8489-94
Khelashvili, George; LeVine, Michael V; Shi, Lei et al. (2013) The membrane protein LeuT in micellar systems: aggregation dynamics and detergent binding to the S2 site. J Am Chem Soc 135:14266-75
Mondal, Sayan; Khelashvili, George; Shi, Lei et al. (2013) The cost of living in the membrane: a case study of hydrophobic mismatch for the multi-segment protein LeuT. Chem Phys Lipids 169:27-38
Quick, Matthias; Shi, Lei; Zehnpfennig, Britta et al. (2012) Experimental conditions can obscure the second high-affinity site in LeuT. Nat Struct Mol Biol 19:207-11
Shan, Jufang; Javitch, Jonathan A; Shi, Lei et al. (2011) The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter. PLoS One 6:e16350
Zhao, Yongfang; Terry, Daniel S; Shi, Lei et al. (2011) Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474:109-13

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