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Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer.
Adhikary, Suraj; Deredge, Daniel J; Nagarajan, Anu; Forrest, Lucy R; Wintrode, Patrick L; Singh, Satinder K.
Afiliação
  • Adhikary S; Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520.
  • Deredge DJ; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201.
  • Nagarajan A; Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
  • Forrest LR; Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
  • Wintrode PL; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201.
  • Singh SK; Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; satinder.k.singh@yale.edu.
Proc Natl Acad Sci U S A ; 114(10): E1786-E1795, 2017 03 07.
Article em En | MEDLINE | ID: mdl-28223522
ABSTRACT
Neurotransmittersodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule 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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Dopamina / Serotonina / Neurotransmissores / Proteínas Cotransportadoras de Sódio-Fosfato Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Dopamina / Serotonina / Neurotransmissores / Proteínas Cotransportadoras de Sódio-Fosfato Idioma: En Ano de publicação: 2017 Tipo de documento: Article