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Measuring cation transport by Na,K- and H,K-ATPase in Xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays.
Zitatschlüssel Durr2013
Autor Katharina L Dürr and Neslihan N Tavraz and Susan Spiller and Thomas Friedrich
Seiten e50201
Jahr 2013
DOI 10.3791/50201
Journal J Vis Exp
Nummer 72
Institution Institute of Chemistry, Technical University of Berlin, Germany.
Zusammenfassung Whereas cation transport by the electrogenic membrane transporter Na(+),K(+)-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H(+),K(+)-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb(+) or Li(+) transport by Na(+),K(+)- or gastric H(+),K(+)-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb(+) (Li(+)) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb(+) uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na(+)/2K(+) transport stoichiometry of the Na(+),K(+)-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H(+),K(+)-ATPase. In principle, the assay is not limited to K(+)-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
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