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

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18 September 2015

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

Billesbølle CB, Krüger MB, Shi L, Quick M, Li Z, Stolzenberg S, Kniazeff J, Gotfryd K, Mortensen JS, Javitch JA, Weinstein H, Loland CJGether UJ Biol Chem. (2015) doi: 10.1074/jbc.M115.677658

A contribution to the transporter field from the Department of Neuroscience and Pharmacology

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 use 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 (TM) 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of TM5 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.

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