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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

Published: February 19th, 2013



1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

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.

We wanted to develop a sensitive, safe and inexpensive alternative to radioactive tracer experiments to investigate the specific transport activity of ion translocating membrane proteins in order to circumvent restrictions regarding the access to isotope laboratories, safety requirements or the use of costly radioisotopes, which - as in the case of lithium - may even be unavailable due to extremely short decay times. We were particularly interested in determining the activity of the electroneutrally operating gastric H+,K+-ATPase, because the enzyme does not generate current and its activity can therefore not be addressed by electrophysiological ....

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1. cDNA Constructs and Protein Expression in Xenopus Oocytes

The cDNA of the membrane protein of interest should be cloned into a vector suitable for expression in Xenopus laevis oocytes such as pTLN9 or pcDNA3.1X10. Such optimized vectors contain the 5'- and 3'-untranslated regions (UTR) of the Xenopus laevis β-globin gene flanking the multiple cloning site (MCS), an RNA polymerase promoter sequence (pTLN: SP6, pcDNA3.1X: T7) located before .......

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Quantification of Rb+ uptake by K+- (or Rb+)-countertransporting P-type ATPases by AAS in the Xenopus oocyte expression system permits reliable determination of enzyme kinetic parameters.

Determination of the transport stoichiometry of the Na+/K+-ATPase

For the electrogenic Na+/K+-ATPase, Rb+ fluxes can be determined in two-electrode voltage-clamp experiments aimed at the c.......

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The described method to measure the amount of Rb+ (or Li+) taken up into individual Xenopus oocytes expressing Na+,K+- or H+,K+-ATPase has proven to be a versatile, flexible and accurate technique to determine the kinetic or thermodynamic parameters of transport for cation-countertransporting P-type ATPases1,4,5,7,8. It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimen.......

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The authors thank Ernst Bamberg (Max-Planck-Institute of Biophysics, Frankfurt, Germany) for generous support during the initial phase of method development, Kazuhiro Abe (Kyoto University, Japan) for numerous fruitful discussions and Dr. Michael Kohl (Analytik-Service, Woltersdorf, Germany) for technical support. The authors gratefully acknowledge funding by the German Research Foundation DFG (Cluster of Excellence "Unifying Concepts in Catalysis"), which also financed the Perkin Elmer AAnalyst 800 apparatus (SFB 498).


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Name Company Catalog Number Comments
Name of the Reagent Company Catalogue Number Comments (optional)
4.0 Ethicon Vicryl suture material Johnson & Johnson V633H
Collagenase type 1A from Clostridium hystolyticum Sigma Aldrich C9891
High-Pure PCR Product Purification Kit Roche Applied Science 11732676001
Nuclease-free water Ambion AM9937
mMessage mMachine Kit SP6/T7 Ambion 1340, 1344
Ouabain octahydrate Sigma Aldrich O3125
Tricain (ethyl 3-aminobenzoate methanesulfonate salt) Sigma Aldrich A5040
Trypsin inhibitor type III-O from chicken egg white Sigma Aldrich T2011
SCH28080 Sigma Aldrich S4443
AAnalyst 800 Perkin Elmer 0993-5256
WinLab32TM Perkin Elmer
BioPhotometer Eppendorf 6131 000.012
Borosilicate Capillaries Science Products GB150F-8P
Hematocrit tubes 3.5" Drummond Scientific 3 000-203-G/X
Hollow Cathode Lamp Lithium Photron P929LL
Hollow Cathode Lamp Rubidium Photron P945
Micropipette Puller Narishige Model PC-10
Oocyte Recording Chamber RC-10 Warner Instr. W4 64-0306
Nanoject II Injection Pump Drummond Scientific 3-000-204
pCLAMP software Molecular Devices
Polypropylene Sample Cup (1.2 ml) Perkin Elmer B0510397
Speedvac - Concentrator model 5301 Eppendorf 5301 000.210
THGA Tube Perkin Elmer B3000641
Turbo TEC-10CX Amplifier NPI Electronics TEC-10CX

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