Format

Send to

Choose Destination
J Biol Chem. 2015 Oct 30;290(44):26725-38. doi: 10.1074/jbc.M115.677658. Epub 2015 Sep 11.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Author information

1
From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
2
Department of Physiology and Biophysics and The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, New York 10065.
3
Center for Molecular Recognition and Departments of Psychiatry and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032.
4
Department of Physiology and Biophysics and.
5
Center for Molecular Recognition and Departments of Psychiatry and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032 Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, and.
6
From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark, gether@sund.ku.dk.

Abstract

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

KEYWORDS:

amino acid transport; conformational change; dopamine transporter; fluorescence quenching; fluorescence spectroscopy; gating; mechanisms of membrane transport; membrane protein; monoamine transporter; neurotransmitter transport

PMID:
26363074
PMCID:
PMC4646326
DOI:
10.1074/jbc.M115.677658
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center