01.07.2021 - SSM-electrophysiology as a Tool for Uncovering Transporter Function
Uncovering the substrates and mechanism of a transporter or demonstrating the functionality of a mutant or variant is a crucial aspect of comprehensive transporter research. In session three of the Transporter Webinar Series, Olga Boudker talks about structural studies on a glutamate transporter and how SSM-based electrophysiology supported her work. From Lars Jeuken we hear about the electrophysiological characterization of a metal transporter.
Title: Cryo-EM structures of human neuronal glutamate transporter
Olga Boudker, Professor of Physiology and Biophysics
(Weill Cornell Medical College)
Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. We have determined cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.
Title: SSM-based electrophysiological characterization of a metal transporter
Lars Jeuken, Professor of Molecular Biophysics,
(Faculty of Biological Sciences, University of Leeds)
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterized using metal-sensitive fluorescent dyes encapsulated in proteoliposomes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with the solid-supported membrane (SSM) technology. SSM uptake assays confirm transport of Mn(II), Co(II), Zn(II), and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) nor Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Although E. faecalis MntH2 is hypothesized to be a proton-metal symporter, no proton symport could be detected with either SSM or fluorescence assays. These data are discussed with respect to fluorescence uptake assays with Mn(II) and Ni(II), where transport was measured on the time scale of minutes, in sharp contrast to the sub-second timescale of the SSM technology.
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