DBP1: Dynamics of Neurotransmitter Transporters: Molecular and Cellular Interactions
PI:Susan Amara, PhD, Scientific Director, Division of Intramural Research Programs, National Institute of Mental Health, NIH; Gonzalo Torres, University of Florida
Funding Source(s): 1ZIAMH002946-03,Structure, function and pharmacology of neurotransmitter reuptake systems (Amara); 7R01 DA038598 Regulation of dopamine transporter function by G protein β−γ subunits (Torres); 9/15/14 - 6/30/19
How the DBP acts as a driver of test bed:
Site-directed mutagenesis, sulfhydryl modification, and chemical cross-linking approaches and biochemical and electro-physiological analyses of EAAT3 provide data for building, testing and refining computational models for anion channeling.
Experiments with cell permeable peptide fragments and DAT mutagenesis, in brain synaptosomes and in vivo drive the computational modeling of DAT - G protein β−γ complex
Data on PKA- and RhoA-dependent signaling events and TAAR1 activation will drive the development and benchmarking of BioNetGen model for identifying pathways/targets that involved in AMPH action on DAT.
Motivation/significance - Sodium-coupled neurotransmitter (NT) transporters regulate neurosignaling in the central nervous system (CNS) and prevent neurotoxicity; yet several aspects of their function remain unknown. First, many transporters act as anion (e.g. chloride, Cl) channels. Anion conductance has been suggested to promote electrogenic Glu- uptake and serve as a sensor for regulating the release of additional Glu-. The Amara lab and others found that Cl- channeling is structurally coupled to Glu- transport, but the molecular basis and mechanism of this coupling is to be elucidated. Second, NT transporters also allow for the efflux (or reverse transport) of NT, modulated by regulatory proteins such as G protein βγ subunits (Gβγ) and CaMKII, addictive drugs such as cocaine and amphetamine (AMPH), or lipids. How the transporter structure adapts to allow for NT efflux remains to be understood. Third, recent studies by the Amara lab highlight the key role of intracellular (IC) AMPH in modulate the function and internalization of transporters; it is not clear how protein-protein interactions (PPIs) stimulated by AMPH alter neurosignaling. This DBP aims at shedding light on these questions for two groups of Na+-coupled transporters: glutamate transporters or so-called excitatory amino acid transporters (EAAT1-3) and dopamine transporter (DAT).
Two major neurotransmitter transporters investigated by the MMBioS team : glutamate transporter (left, illustrated here for the archaeal aspartate transporter GltPh) representative of human excitatory amino acid transporters) and dopamine transporter (right; representative of NSS family members sharing the LeuT fold).
This project aims to determine the mechanism of substrate binding and release and ion permeation in neurotransmitter transporters. Our continuing efforts to use cysteine modification and crosslinking and a variety of other mutational approaches to probe structure-function relationships should provide a powerful resource with which to experimentally test hypotheses generated from computational modeling approaches.Experiments will be performed on the relevant human transporters including site-directed mutagenesis, biochemical labeling and whole cell voltage-clamp recording. We have two specific aims:
To determine the substrate and ion binding, occlusion, and release mechanisms in excitatory amino acid transporters (EAATs)
A recent high-resolution structure of GltPh suggests that the core translocation domain is capable of a large downward motion perpendicular to the membrane. Recent data from our lab describes an inward shift of one of the monomers when the other two subunits within the trimer are crosslinked. Additionally, we have found a point mutation, R388D, which shifts the core domain inward. Interestingly, this mutation also abolishes the substrate gating of the anion channel suggesting a role for core domain movement in anion channel function. We are examining a series of cysteine mutants that when cross-linked or modified may restrict movements of different domains in EAAT1.
To identify the pathways for uncoupled anion permeation through EAATs
Analysis of chloride flux supports the idea that prokaryotic transporters like their eukaryotic counterparts, also permeate chloride and other anions. To aid in these efforts, the Bahar laboratory has undertaken a series of molecular dynamics (MD) simulations and elastic network model (ENM) analyses, examining interactions of chloride with the IC and extracellular (EC) sides of the carrier. By combining computation data on anion channel gating and permeation with biophysical and biochemical data, we will be able to gain unique insight in the molecular mechanisms underlying these carrier functions.