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).

<ol>
	<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=Impaired+plasma+membrane+targeting+or+protein+stability+by+certain+ATP1A2+mutations+identified+in+sporadic+or+familial+hemiplegic+migraine.">Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine.</a> Channels (Austin). 3, 82-87 (2009).</li><li>Castillo, 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=Energy+landscape+of+the+reactions+governing+the+Na++deeply+occluded+state+of+the+Na+/K+-ATPase+in+the+giant+axon+of+the+Humboldt+squid.">Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.</a> Proc. Natl. Acad. Sci. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromultimeric+CLC+chloride+channels+with+novel+properties.">Heteromultimeric CLC chloride channels with novel properties.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).</li><li>Koenderink, J. B., 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=Na,K-ATPase+mutations+in+familial+hemiplegic+migraine+lead+to+functional+inactivation.">Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation.</a> Biochim. Biophys. Acta. 1669, 61-68 (2005).</li><li>Price, E. M. 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. Physiol. 136, 63-82 (2010).</li><li>Chakavarti, B., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+separation+of+proteins.">Electrophoretic separation of proteins.</a> J. Vis. Exp. (16), e758 (2008).</li><li>Kamsteeg, E. J., Deen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+aquaporin-2+expression+levels+in+genotype+-phenotype+studies+in+nephrogenic+diabetes+insipidus.">Importance of aquaporin-2 expression levels in genotype -phenotype studies in nephrogenic diabetes insipidus.</a> Am. J. Physiol. Renal Physiol. 279, 778-784 (2000).</li><li>Koenderink, J. B., 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=Electrophysiological+analysis+of+the+mutated+Na,K-ATPase+cation+binding+pocket.">Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.</a> J. Biol. Chem. 278, 51213-51222 (2003).</li><li>Gottardi, C. J., Caplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+requirements+for+the+cell-surface+expression+of+multisubunit+ion-transporting+ATPases.+Identification+of+protein+domains+that+participate+in+Na,K-ATPase+and+H,K-ATPase+subunit+assembly.">Molecular requirements for the cell-surface expression of multisubunit ion-transporting ATPases. Identification of protein domains that participate in Na,K-ATPase and H,K-ATPase subunit assembly.</a> J. Biol. Chem. 268, 14342-14347 (1993).</li><li>Mathews, P. M., 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=Primary+structure+and+functional+expression+of+the+mouse+and+frog+alpha-subunit+of+the+gastric+H+-K+-ATPase.">Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H+-K+-ATPase.</a> Am. J. Physiol. 268, 1207-1214 (1995).</li><li>Horisberger, J. D., 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+H,K-ATPase+beta-subunit+can+act+as+a+surrogate+for+the+beta-subunit+of+Na,K-pumps.">The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.</a> J. Biol. Chem. 266, 19131-19134 (1991).</li><li>Guennoun-Lehmann, S., Fonseca, J. E., Horisberger, J. D., Rakowski, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palytoxin+acts+on+Na+,K+-ATPase+but+not+nongastric+H+,K+-ATPase.">Palytoxin acts on Na+,K+-ATPase but not nongastric H+,K+-ATPase.</a> J. Membr. Biol. 216, 107-116 (2007).</li><li>Horisberger, J. D., Guennoun, S., Burnay, M., Geering, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation+stoichiometry+and+cation+pathway+in+the+Na,K-ATPase+and+nongastric+H,K-ATPase.">Cation stoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase.</a> Ann. N.Y. Acad. Sci.. 986, 127-132 (2003).</li></ol>

The authors declare that they have no competing financial interests.

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.

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

Mess-Cation Transport von Na, K-und H, K-ATPase in<em&gt; Xenopus</em&gt; Oozyten mittels Atomabsorptionsspektrometrie: Eine Alternative zu Radioisotopen Assays

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

measuring-cation-transport-nak-hk-atpase-xenopus-oocytes-atomic

Research

JoVE Journal

Biology

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

10.5K Aufrufe.  Technical University of Berlin. 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.

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

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Spannklemme Fluorometrie in<em&gt; Xenopus</em&gt; Oozyten mit fluoreszierenden unnatürlichen Aminosäuren

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Forschung

Biochemie

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Messung der Trans-Plasma Membran Elektronentransport durch C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Reinigung und Wiederherstellung der TRPV1 für spektroskopische Analyse

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

Eine semi-High-Throughput-Anpassung des NADH-gekoppelten ATPase-Assays zum Screening kleiner Molekülinhibitoren

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Messung der Nukleotidbindung an intakte, funktionelle Membranproteine in Echtzeit

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

Untersuchung der Funktionen und Aktivitäten des neuronalen K-Cl-Co-Transporters KCC2 mittels Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Demonstration der proteolytischen Aktivierung des epithelialen Natriumkanals (ENaC) durch Kombination von Strommessungen mit Nachweis der Spaltfragmente

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Biologie

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Funktionelle Charakterisierung von Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Wärmetauscher von intrazellulären Kompartimente Mit Proton-Tötung Selection man sie an der Plasmamembran Express

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Die Messung der extrazellulären Ionenflüsse Verwendung der ionenselektiven selbst verweisMikroElektroden-Technik

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Die Nutzung der Ramsay Assay zu Flüssigkeitssekretion und Ionenfluss Preise in der Maßnahme<em&gt; Drosophila melanogaster</em&gt; Malpighischen Tubulus

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Mess-Cation Transport von Na, K-und H, K-ATPase in

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Spannklemme Fluorometrie in