Single-molecule dynamics of gating in a neurotransmitter transporter homologue.
Neurotransmitter:Na(+) symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na(+) gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.
Zhao
Yongfang
Y
Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, New York 10032, USA.
Terry
Daniel
D
Shi
Lei
L
Weinstein
Harel
H
Blanchard
Scott C
SC
Javitch
Jonathan A
JA
D000409:Alanine / Q000378:metabolism; D000494:Allosteric Regulation; D029063:Aquifoliaceae / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer; D007930:Leucine / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism
D001426:Bacterial Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D012964:Sodium; D007930:Leucine; D003545:Cysteine; D000409:Alanine
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