Mätning Cation Transport med Na, K-och H, K-ATPas i

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Dürr, K. L., Tavraz, N. N., Spiller, S., Friedrich, T. Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays. J. Vis. Exp. (72), e50201, doi:10.3791/50201 (2013).

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Abstract

Materials

Name Company Catalog Number Comments
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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References

  1. Tavraz, N. N., et al. Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine. Channels (Austin). 3, 82-87 (2009).
  2. Castillo, J. P., et al. Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid. Proc. Natl. Acad. Sci. U.S.A. 108, 20556-20561 (2011).
  3. Friedrich, T., Bamberg, E., Nagel, G. Na+,K+-ATPase pump currents in giant excised patches activated by an ATP concentration jump. Biophys J. 71, 2486-2500 (1996).
  4. Dürr, K. L., Tavraz, N. N., Dempski, R. E., Bamberg, E., Friedrich, T. Functional significance of E2 state stabilization by specific alpha/beta-subunit interactions of Na,K- and H,K-ATPase. J. Biol. Chem. 284, 3842-3854 (2009).
  5. Dürr, K. L., Abe, K., Tavraz, N. N., Friedrich, T. 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. J. Biol. Chem. 284, 20147-20154 (2009).
  6. Durr, K. L., Tavraz, N. N., Zimmermann, D., Bamberg, E., Friedrich, T. Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes. Biochemistry. 47, 4288-4297 (2008).
  7. Dürr, K. L., Tavraz, N. N., Friedrich, T. Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes. PLoS One. 7, e33645 (2012).
  8. Tavraz, N. N., et al. Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2. J. Biol. Chem. 283, 31097-31106 (2008).
  9. Lorenz, C., Pusch, M., Jentsch, T. J. Heteromultimeric CLC chloride channels with novel properties. Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).
  10. Koenderink, J. B., et al. Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation. Biochim. Biophys. Acta. 1669, 61-68 (2005).
  11. Price, E. M. J., Lingrel, B. 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. Biochemistry. 27, 8400-8408 (1988).
  12. Richards, R., Dempski, R. E. Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling. J. Vis. Exp. (2627), e2627 (2011).
  13. Lafaire, A. V., Schwarz, W. Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis. J. Membr. Biol. 91, 43-51 (1986).
  14. Morth, J. P., Pedersen, B. P., Toustrup-Jensen, M. S., Sorensen, T. L., Petersen, J., Andersen, J. P., Vilsen, B., Nissen, P. Crystal structure of the sodium-potassium pump. Nature. 450, 1043-1049 (2007).
  15. Toustrup-Jensen, M. S., et al. The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935. J. Biol. Chem. 284, 18715-18725 (2009).
  16. Morth, J. P., et al. The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364, 217-227 (2009).
  17. Yaragatupalli, S., Olivera, J. F., Gatto, C., Artigas, P. Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump. Proc. Natl. Acad. Sci. U.S.A. 106, 15507-15512 (2009).
  18. Meier, S., Tavraz, N. N., Durr, K. L., Friedrich, T. Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit. J. Gen. Physiol. 135, 115-134 (2010).
  19. Vedovato, N., Gadsby, D. C. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions. J. Gen. Physiol. 136, 63-82 (2010).
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  22. Koenderink, J. B., et al. Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket. J. Biol. Chem. 278, 51213-51222 (2003).
  23. Gottardi, C. J., Caplan, M. J. 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. J. Biol. Chem. 268, 14342-14347 (1993).
  24. Mathews, P. M., et al. Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H+-K+-ATPase. Am. J. Physiol. 268, 1207-1214 (1995).
  25. Horisberger, J. D., et al. The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps. J. Biol. Chem. 266, 19131-19134 (1991).
  26. Guennoun-Lehmann, S., Fonseca, J. E., Horisberger, J. D., Rakowski, R. F. Palytoxin acts on Na+,K+-ATPase but not nongastric H+,K+-ATPase. J. Membr. Biol. 216, 107-116 (2007).
  27. Horisberger, J. D., Guennoun, S., Burnay, M., Geering, K. Cation stoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase. Ann. N.Y. Acad. Sci.. 986, 127-132 (2003).

Comments

2 Comments

  1. Your video is very helpful for me, but I can not see the whole film in CHINA. Could you help me?

    Reply
    Posted by: wei l.
    April 8, 2013 - 12:38 PM
  2. I am sorry, but I am not allowed to distribute the video, unfortunately, we could not cover the costs for full open access of the material. But I will answer any questions related to the protocol, if you contact me directly (friedrich@chem.tu-berlin.de)

    Reply
    Posted by: Thomas F.
    April 9, 2013 - 8:54 AM

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