This was the fifth year for this project, which is using biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimers disease, and exitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model glutamate transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We previously discovered that the bacterial glutamate transporters display a chloride transport activity which is stoichiometrically uncoupled from glutamate uptake. This chloride transport activity is similar to one which is important in the mammalian transporters and suggests that the bacterial homologs provide an excellent structural model in which to study the process of chloride transport in these proteins. Our studies on conformational changes in these proteins continued this year;using limited proteolysis and cysteine scanning mutagenesis, we found evidence of a significant substrate-induced conformational change and localized the change to a novel protein heretofore not expected to participate in the transport process. We further found that the protein movement is an essential part of the transport process. Also, we are using EPR spectroscopy to probe transport-related conformational changes and have found substantial evidence for major movements of the protein, which oppose and separate pairs of spin labels with changes in substrate and sodium. We are currently exploring the extent of these changes and their kinetics.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2011
Total Cost
$659,396
Indirect Cost
City
State
Country
Zip Code
Shlosman, Irina; Marinelli, Fabrizio; Faraldo-Gómez, José D et al. (2018) The prokaryotic Na+/Ca2+ exchanger NCX_Mj transports Na+ and Ca2+ in a 3:1 stoichiometry. J Gen Physiol 150:51-65
Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A (2017) A general method for determining secondary active transporter substrate stoichiometry. Elife 6:
Mulligan, Christopher; Mindell, Joseph A (2017) Pinning Down the Mechanism of Transport: Probing the Structure and Function of Transporters Using Cysteine Cross-Linking and Site-Specific Labeling. Methods Enzymol 594:165-202
Mulligan, Christopher; Fenollar-Ferrer, Cristina; Fitzgerald, Gabriel A et al. (2016) The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism. Nat Struct Mol Biol 23:256-63
Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Mulligan, Christopher et al. (2015) Family resemblances: A common fold for some dimeric ion-coupled secondary transporters. J Gen Physiol 146:423-34
Mulligan, Christopher; Fitzgerald, Gabriel A; Wang, Da-Neng et al. (2014) Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J Gen Physiol 143:745-59
Parker, Joanne L; Mindell, Joseph A; Newstead, Simon (2014) Thermodynamic evidence for a dual transport mechanism in a POT peptide transporter. Elife 3:
Mulligan, Christopher; Mindell, Joseph A (2013) Mechanism of transport modulation by an extracellular loop in an archaeal excitatory amino acid transporter (EAAT) homolog. J Biol Chem 288:35266-76
Compton, Emma L R; Taylor, Erin M; Mindell, Joseph A (2010) The 3-4 loop of an archaeal glutamate transporter homolog experiences ligand-induced structural changes and is essential for transport. Proc Natl Acad Sci U S A 107:12840-5
Knepper, Mark A; Mindell, Joseph A (2009) Structural biology: Molecular coin slots for urea. Nature 462:733-4

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