Fluorometric Characterization of Substrate Binding in a Bacterial NSS Homologue

Abstract

The prokaryotic amino acid transporter LeuT belongs to the neurotransmitter:sodium symporter (NSS) family that also includes mammalian transporters of neurotransmitters such as dopamine, serotonin and nor-epinephrine. NSS are key terminators of synaptic transmission in CNS, and are the targets of various therapeutics as well as drugs of abuse. Here we seek to further elucidate the molecular details of the sodium dependent translocation cycle by which substrate is transported across the cell membrane. To this end, we have introduced the environmentally sensitive fluorophore tetramethylrhodamine to the cytosolic end of TM5 in LeuT a region shown in mammalian NSS to undergo change in solvent accessibility during substrate translocation. Fluorescent labelling of the single cystein mutant yields a functional transporter. With fluorescence spectroscopy, we then demonstrate that binding of a transported substrate induces a sodium dependent increase in accessibility of the fluorophore to the hydrophilic quencher iodide (KI). Tryptophan, a competitive inhibitor of WT LeuT, only induces an increase in fluorophore accessibility when the change of substrate specificity mutation I359Q is introduced. Finally, addition of the tricyclic antidepressant clomipramine (CMI) only increases the apparent accessibility in the presence of a transported substrate. Collectively, our findings represent a direct measure of substrate bound occluded state formation, a conformational step preceding transporter isomerization to the inward facing conformation.