Conformational dynamics of a neurotransmitter: Sodium symporter in a lipid bilayer
JournalProceedings of the National Academy of Sciences of the United States of America
PublisherNational Academy of Sciences
MetadataShow full item record
AbstractNeurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of smallmolecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.
SponsorsThis work was supported by the Alfred P. Sloan Foundation (S.K.S.); the Brain and Behavior Research Foundation (S.K.S.); a Goodman- Gilman Yale Scholar Award (to S.K.S.); NIH/National Institute of Mental Health Grants R00MH083050 (to S.K.S.) and R01MH100688 (to S.K.S.); the Division of Intramural Research of the NIH, National Institute of Neurological Disorders and Stroke (L.R.F.); and the University of Maryland Baltimore, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014).
Hydrogen-deuterium exchange mass spectrometry
Molecular dynamics simulations
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85014862382&doi=10.1073%2fpnas.1613293114&partnerID=40&md5=597f3dbe0c24a5b20edb9cbf398c202f; http://hdl.handle.net/10713/11353
- LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake.
- Authors: Zhou Z, Zhen J, Karpowich NK, Goetz RM, Law CJ, Reith ME, Wang DN
- Issue date: 2007 Sep 7
- Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS.
- Authors: Möller IR, Merkle PS, Calugareanu D, Comamala G, Schmidt SG, Loland CJ, Rand KD
- Issue date: 2020 Aug 15
- Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.
- Authors: Billesbølle CB, Krüger MB, Shi L, Quick M, Li Z, Stolzenberg S, Kniazeff J, Gotfryd K, Mortensen JS, Javitch JA, Weinstein H, Loland CJ, Gether U
- Issue date: 2015 Oct 30
- Neurotransmitter transporters in schistosomes: structure, function and prospects for drug discovery.
- Authors: Ribeiro P, Patocka N
- Issue date: 2013 Dec
- Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters.
- Authors: Stolzenberg S, Quick M, Zhao C, Gotfryd K, Khelashvili G, Gether U, Loland CJ, Javitch JA, Noskov S, Weinstein H, Shi L
- Issue date: 2015 May 29