Name: measuring cation transport by na,k- and h,k-atpase in xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays
Uploaded: 2013-02-19T12:00:00
Description: Watch this Scientific Journal Video about Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays at JoVE.co

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).

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 &beta;-globin gene flanking the multiple cloning site (MCS), an RNA polymerase promoter sequence (pTLN: SP6, pcDNA3.1X: T7) located before ...

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U.S.A. 108, 20556-20561 (2011).</li><li>Friedrich, T., Bamberg, E., Nagel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+,K+-ATPase+pump+currents+in+giant+excised+patches+activated+by+an+ATP+concentration+jump.">Na+,K+-ATPase pump currents in giant excised patches activated by an ATP concentration jump.</a> Biophys J. 71, 2486-2500 (1996).</li><li>D&#252;rr, K. L., Tavraz, N. N., Dempski, R. E., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+E2+state+stabilization+by+specific+alpha/beta-subunit+interactions+of+Na,K-+and++H,K-ATPase.">Functional significance of E2 state stabilization by specific alpha/beta-subunit interactions of Na,K- and H,K-ATPase.</a> J. Biol. Chem. 284, 3842-3854 (2009).</li><li>D&#252;rr, K. L., Abe, K., Tavraz, N. N., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E2P+state+stabilization+by+the+N-terminal+tail+of+the+H,K-ATPase+beta-subunit+is+critical+for+efficient+proton+pumping+under+in+vivo+conditions.">E2P state stabilization by the N-terminal tail of the H,K-ATPase beta-subunit is critical for efficient proton pumping under in vivo conditions.</a> J. Biol. Chem. 284, 20147-20154 (2009).</li><li>Durr, K. L., Tavraz, N. N., Zimmermann, D., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Na,K-ATPase+and+H,K-ATPase+enzymes+with+glycosylation-deficient+beta-subunit+variants+by+voltage-clamp+fluorometry+in+Xenopus+oocytes.">Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes.</a> Biochemistry. 47, 4288-4297 (2008).</li><li>D&#252;rr, K. L., Tavraz, N. N., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+gastric+H,K-ATPase+activity+by+cations,+voltage+and+intracellular+pH+analyzed+by+voltage+clamp+fluorometry+in+Xenopus+oocytes.">Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes.</a> PLoS One. 7, e33645 (2012).</li><li>Tavraz, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+functional+consequences+of+mutations+in+the+Na+/K+-ATPase+alpha2-subunit+causing+familial+hemiplegic+migraine+type+2.">Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.</a> J. Biol. Chem. 283, 31097-31106 (2008).</li><li>Lorenz, C., Pusch, M., Jentsch, T. 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J., Lingrel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+in+the+Na,K-ATPase+alpha+subunit:+site-directed+mutagenesis+of+glutamine-111+to+arginine+and+asparagine-122+to+aspartic+acid+generates+a+ouabain-resistant+enzyme.">Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.</a> Biochemistry. 27, 8400-8408 (1988).</li><li>Richards, R., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+conformational+dynamics+of+membrane+proteins+in+situ+with+site-directed+fluorescence+labeling.">Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.</a> J. Vis. Exp. (2627), e2627 (2011).</li><li>Lafaire, A. V., Schwarz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependence+of+the+rheogenic+Na+/K++ATPase+in+the+membrane+of+oocytes+of+Xenopus+laevis.">Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis.</a> J. Membr. Biol. 91, 43-51 (1986).</li><li>Morth, J. P., Pedersen, B. P., Toustrup-Jensen, M. S., Sorensen, T. L., Petersen, J., Andersen, J. P., Vilsen, B., Nissen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+sodium-potassium+pump.">Crystal structure of the sodium-potassium pump.</a> Nature. 450, 1043-1049 (2007).</li><li>Toustrup-Jensen, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C+terminus+of+Na+,K+-ATPase+controls+Na++affinity+on+both+sides+of+the+membrane+through+Arg935.">The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935.</a> J. Biol. Chem. 284, 18715-18725 (2009).</li><li>Morth, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+Na+,K+-ATPase+and+mapping+of+isoform+differences+and+disease-related+mutations.">The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364, 217-227 (2009).</li><li>Yaragatupalli, S., Olivera, J. F., Gatto, C., Artigas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Na++transport+after+an+intracellular+alpha-subunit+deletion+reveals+strict+external+sequential+release+of+Na++from+the+Na/K+pump.">Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 15507-15512 (2009).</li><li>Meier, S., Tavraz, N. N., Durr, K. L., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+inward+leakage+currents+caused+by+deletion+or+mutation+of+carboxy-terminal+tyrosines+of+the+Na+/K+-ATPase+{alpha}+subunit.">Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.</a> J. Gen. Physiol. 135, 115-134 (2010).</li><li>Vedovato, N., Gadsby, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+C-terminal+tyrosines+stabilize+occluded+Na/K+pump+conformations+containing+Na+or+K+ions.">The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.</a> J. Gen. 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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...

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 methods. Since Na+,K+- and H+,K+-ATPase transport Rb+ as efficient as K+ (and Li+ as well), the high sensitivity of the AAS technique for rubidium or lithium should facilitate sensitive detection of transport activity. Atomic absorption spectrophotometers are common analytical devices, which are widely distributed in chemical laboratories and should be accessible to a large number of interested scientists. Furthermore, we wanted to take advantage of the Xenopus oocyte expression system, which utilizes large single cells (about 1.0-1.5 mm diameter) that allow to achieve a remarkably low cell-to-cell variability regarding the protein expression level within a single batch. A simple calculation demonstrates the feasibility of the AAS assay: The detection limit (characteristic mass) for rubidium with the THGA-AAS technique is 10 pg or 1.2&middot;10-13 mol (Rb: 85.47 g/mol), for lithium 5.5 pg or 7.9&middot;10-13 mol (Li: 6.94 g/mol). Upon heterologous expression of Na+,K+-ATPase in Xenopus oocytes, pump currents of 100 nA can be achieved (which equals about 6.2&middot;1011 elementary charges per second, or 1.03&middot;10-12 mol s-1), thus resulting in a transport of 6&middot;10-6 C of charge within 1 min. Since the transport of one net charge corresponds to the uptake of two Rb+ ions (due to the 3Na+/2K+ stoichiometry), 100 nA current for 1 min corresponds to an uptake of 1.2&middot;10-10 mol Rb+. Thus, even upon a 1,000-fold dilution (homogenization of an oocyte with about 1 &mu;l volume in 1 ml water), a typical THGA-AAS sample (20 &mu;l) contains 2.4&middot;10-12 mol Rb+ (or 204 pg), which is far above the detection threshold. Therefore, even transporters with more than 100-fold lower transport activity or plasma membrane expression can be assayed with the technique by appropriately adjusting the flux time of the experiment.
Since the pumping rate is sensitively dependent on temperature (typical activation energies for Na+,K+-ATPase are in the range of 90 kJ/mol to 130 kJ/mol1-3, which results in an about 30% increase in the turnover rate upon a change from 20 &deg;C to 22 &deg;C), it is mandatory to carry out the flux measurements under precise temperature control (air conditioning) with well equilibrated buffer solutions. Furthermore, oocytes should be carefully selected regarding homogenous size for the expression of an ion transporter. With these precautions, it is possible to routinely achieve experimental standard errors of less than 10 percent with about 10 cells per experimental condition. Using this technique, we were able to determine e.g. the apparent Rb+ affinities of cation transport4-6, the influence of extra- and intracellular pH7 and the effect of mutations of residues involved in cation coordination during transport4,8. An advantage of the technique is that ion fluxes can also be determined in combination with two-electrode voltage clamping of the oocytes, which on one hand assures accurate control of the membrane potential during transport and on the other hand allows to correlate ion flux with membrane current. Thus, it was possible to verify the 3Na+/2K+ stoichiometry of the Na+,K+-ATPase (see exemplary results below) and to determine the voltage dependence of cation transport of the gastric H+/K+-ATPase7.

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 <td>4.0 Ethicon Vicryl 			suture material</td>
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 <td>Roche Applied Science</td>
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 <td>mMessage mMachine Kit SP6/T7</td>
 <td>Ambion</td>
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 <td>Ouabain octahydrate</td>
 <td>Sigma Aldrich</td>
 <td>O3125</td>
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 <td>Tricain (ethyl 3-aminobenzoate 			methanesulfonate salt)</td>
 <td>Sigma Aldrich</td>
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 <td>Drummond Scientific</td>
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 <td>Turbo TEC-10CX Amplifier</td>
 <td>NPI Electronics</td>
 <td>TEC-10CX</td>
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 </tbody>
</table>

measuring cation transport by na,k- and h,k-atpase in xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays

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.

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...

Watch this Scientific Journal Video about Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays at JoVE.co

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

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.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...