Single-molecule dynamics of gating in a neurotransmitter transporter homolog

Neurotransmitter:Na(+) symporters (NSS) remove neurotransmitters from the synapse in a reuptake process driven by the Na(+) gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behavior and mood. In order to probe the nature of conformational changes associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay, in combination with functional and computational studies, using the prokaryotic NSS homolog 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 and/or transport. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that may be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism coupling ion and substrate binding to transport.