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Gewählte Publikation:

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Publikationstyp: Zeitschriftenaufsatz
Dokumentart: Originalarbeit

Publikationsjahr: 2019

AutorInnen: Kreiter, J; Pohl, EE

Titel: A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins.

Quelle: J Vis Exp. 2019 (143), e58552.



Autor/innen der Vetmeduni Vienna:

Kreiter Jürgen
Pohl Elena

Beteiligte Vetmed-Organisationseinheiten
Institut für Physiologie, Pathophysiologie und Biophysik, Abteilung für Physiologie und Biophysik


Zugehörige(s) Projekt(e): Proton leak through Adenine Nucleotide Translocase


Abstract:
To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of membrane carrier proteins reconstituted in lipid bilayer membranes is a powerful but delicate method for the assessment of their physicochemical and pharmacological properties. The substrate turnover number is a unique parameter that allows the comparison of the activity of different membrane proteins. In an electrogenic transport, the gradient of the translocated substrate creates a membrane potential that directly correlates to the substrate turnover rate of the protein. By using silver chloride electrodes, a diffusion potential, also called liquid junction potential, is induced, which alters electrode potential and significantly disturbs precise membrane potential measurements. Diffusion potential can be minimized by a salt bridge, which balances electrode potential. In this article, a micro-agar salt bridge is designed to improve the electrophysiological set-up, which uses micropipettes for the membrane formation. The salt solution is filled into a microcapillary pipette tip, stabilized by the addition of agarose, and can be easily mounted to a standard electrode. The electrode potential of a micro-salt bridge electrode is more stable compared to a standard electrode. The implementation of this system stabilizes electrode potential and allows more precise measurements of membrane potential generated by a pH gradient. Using this system, the proton turnover rates of the mitochondrial carriers UCP1 and UCP3 are reinvestigated and compared to earlier measurements.


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