Journal Article

Quantifying secondary transport at single-molecule resolution

Blanchard, Scott C., Javitch, Jonathan A., Warren, Audrey L., Terry, Daniel S., Quick, Matthias, Fitzgerald, Gabriel A.

2019

Overview

Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes 1 , 2 . Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters 3 , by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism 4 , 5 , including the rate at which the transporter returns to an outward-facing state after the transported substrate is released. Imaging of substrate transport by individual MhsT transporters, members of the neurotransmitter:sodium symporter family of secondary transporters, at single- and multi-turnover resolution reveals that the rate-limiting step varies with the identity of the transported substrate.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/62 / 631/57/2265 / 631/57/2270/1140 / 96/33 / Allosteric Site / Amino Acids - analysis / Amino Acids - chemistry / Amino Acids - metabolism / Analysis / Bacterial Proteins - analysis / Bacterial Proteins - metabolism / Biological Transport / Cell Survival / Fluorescence Resonance Energy Transfer / Humanities and Social Sciences / Hydrophobic and Hydrophilic Interactions / Ion transport / Kinetics / Lipid Bilayers - metabolism / Measurement / Membrane proteins / multidisciplinary / Neurotransmitters / Observations / Physiological aspects / Protein Conformation / Science / Science (multidisciplinary) / Single Molecule Imaging / Substrates (Biochemistry) / Symporters - analysis / Symporters - chemistry / Symporters - metabolism