Abstract
This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, γa and γb, decrease the apparent affinity of the pump for Na+ and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive 86Rb(K+) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K0.5(Na) at high K+ concentration and a decrease in K′ATP. Comparison of clones of γ-transfected and mock-transfected cells (with similar Vmax values) indicated that γ causes a modest ≈30% increase in the steady-state concentration of intracellular Na+. Furthermore, for both γa and γb, values of intracellular Na+ were similar to those predicted from the kinetic constants, K0.5(Na) and Vmax. Finally, there was a γ-mediated increase in apparent affinity for extracellular K+, which had not been detected in assays of permeabilized membranes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Notes
Appropriateness of using K0.5(Na) from ATPase assays carried out at 100 mM K+ is indicated by data showing that K0.5(Na) values for at least the two isoforms, α1 and α2, are 16.5 ± 0.1 (Table 2) and 21.9 ± 2.25 (A. Zouzoulas and R. Blostein, unpublished data), respectively, which are notably similar to K0.5(Na) determined in flux experiments, i.e., 17.2 ± 1.12 and 19.68 ± 0.96, respectively (values from Munzer et al., 1994).
References
Arystarkhova E., Wetzel R.K., Asinovski N.K., Sweadner K.J. 1999. The γ subunit modulates Na+ and K+ affinity of the renal Na,K-ATPase. J. Biol. Chem. 274:33183–33185
Arystarkhova E., Wetzel R.K., Sweadner K.J. 2002. Distribution and oligomeric association of splice forms of Na,K-ATPase regulatory γ subunit in rat kidney. Am. J. Physiol. 282:F393–F407
Balshaw D.M., Millette L.A., Tepperman K., Wallick E.T. 2000. Combined allosteric and competitive interaction between extracellular Na+ and K+ during ion transport by the α1, α2, and α3 isoforms of the Na, K-ATPase. Biophys. J. 79:853–862
Beguin P., Crambert G., Guennoun S., Garty H., Horisberger J.D., Geering K. 2001. CHIP, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the γ subunit. EMBO J. 20:3993–4002
Cornelius F., Mahmmoud Y.A., Christensen H.R. 2001. Modulation of Na,K-ATPase by associated small transmembrane regulatory proteins and by lipids. J. Bioenerg. Biomembr. 33:415–423
Crambert G., Geering K. 2003. FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. Science’s Stke [Electronic Resource]: Signal Transduction Knowledge Environment. 166:21
Eisner D.A., Richards D.E. 1981. The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts. J. Physiol. 319:403–418
Forbush B., Kaplan J.H., Hoffman J.F. 1978. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase. Biochemistry. 17:3667–3676
Garty H., Lindzen M., Scanzano R., Aizman R., Fuzesi M., Goldshleger R., Farman N., Blostein R., Karlish S.J. 2002. A functional interaction between CHIF and Na-K-ATPase: implication for regulation by FXYD proteins. Am. J. Physiol. 283:F607–F615
Geering K., Beguin P., Garty H., Karlish S., Fuzesi M., Horisberger J.D., Crambert G. 2003. FXYD proteins: new tissue- and isoform-specific regulators of Na,K-ATPase. Ann. N.Y. Acad. Sci. 986:388–394
Hoffman J.F., Wickrema A., Potapova O., Milanick M., Yingst D.R. 2002. Na pump isoforms in human erythroid progenitor cells and mature erythrocytes. Proc. Natl. Acad. Sci. USA. 99:14572–14577
Ikehara T., Yamaguchi H., Hosokawa K., Sakai T., Miyamoto H. 1984. Rb+ influx in response to changes in energy generation: effect of the regulation of the ATP content of HeLa cells. J. Cell. Physiol. 119:273–282
Jewell E.A., Lingrel J.B. 1991. Comparison of the substrate dependence properties of the rat Na,K-ATPase α1, α2, and α3 isoforms expressed in HeLa cells. J. Biol. Chem. 266:16925–16930
Kuster B., Shainskaya A., Pu H.X., Goldshleger R., Blostein R., Mann M., Karlish S J. 2000. A new variant of the y subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells. J. Biol. Chem. 275:18441–18446
Li C., Grosdidier A., Crambert G., Horisberger J.D., Michielin O., Geering K. 2004. Structural and functional interaction sites between Na,K-ATPase and FXYD proteins. J. Biol. Chem. 279:38895–38902
Meij I.C., Koenderink J.B., van Bokhoven H., Assink K.F., Groenestege W.T., de Pont J.J., Bindels R.J., Monnens L.A., van den Heuvel L.P., Knoers N.V. 2000. Dominant isolated renal magnesium loss is caused by misrouting of the Na, K-ATPase γ-subunit. Nature Genetics 26:265–266
Mercer R.W., Biemesderfer D., Bliss D.P., Jr., Collins J.H., Forbush B., 3rd. 1993. Molecular cloning and immunological characterization of the γ polypeptide, a small protein associated with the Na,K-ATPase. J. Cell Biol 121:579–586
Munzer J.S., Daly S.E., Jewell-Motz E.A., Lingrel J.B., Blostein R. 1994. Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase. J. Biol. Chem. 269:16668–16676
Pu H.X., Cluzeaud F., Goldshleger R., Karlish S.J., Farman N., Blostein R. 2001. Functional role and immunocytochemical localization of the γa and γb forms of the Na,K-ATPase γ subunit. J. Biol. Chem. 276:20370–20378
Pu H.X., Scanzano R., Blostein R. 2002. Distinct regulatory effects of the Na,K-ATPase γ subunit. J. Biol. Chem. 277:20270–20276
Soltoff S.P., Mandel L.J. 1984. Active ion transport in the renal proximal tubule. III. The ATP dependence of the Napump. J. Gen. Physiol. 84:643–662
Sweadner K.J., Rael E. 2000. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68:41–56
Therien A.G., Blostein R. 1999. K/Na antagonism at cytoplasmic sites of Na, K-ATPase: a tissue-specific mechanism of sodium pump regulation. Am. J. Physiol. 277:C891–C898
Therien A.G., Blostein R. 2000. Mechanisms of sodium pump regulation. Am. J. Physiol. 279:C541–566
Therien A.G., Goldshleger R., Karlish S.J., Blostein R. 1997. Tissue-specific distribution and modulatory role of the γ subunit of the Na,K-ATPase. J. Biol. Chem. 272:32628–32634
Therien A.G., Karlish S.J., Blostein R. 1999. Expression and functional role of the γ subunit of the Na, K-ATPase in mammalian cells. J. Biol. Chem. 274:12252–12256
Therien A.G., Pu H.X., Karlish S.J., Blostein R. 2001. Molecular and functional studies of the γ subunit of the sodium pump. J. Bioenerg. Biomembr. 33:407–414
Therien A.G., Pu H.X., Karlish S.J.D., Blostein R. 2000. Structure/function studies of the γ subunit of renal Na,K-ATPase. In: K. Taniguchi and S. Kaya, editors. Na/K-ATPase and Related ATPases. pp. 481–488. Elsevier, Sapporo, Japan
Wetzel R.K., Sweadner K.J. 2001. Immunocytochemical localization of Na-K-ATPase α- and γ-subunits in rat kidney. Am. J. Physiol. 281:F531–F545
Acknowledgements
We thank Dr. J. B. Lingrel for the rat α1-transfected HeLa cells. This work was supported by grant MT-3876 from the Canadian Institutes for Health research (to RB) and from the NIDDK R37 DK-33640 (to PBD).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zouzoulas, A., Dunham, P. & Blostein, R. The Effect of the Gamma Modulator on Na/K Pump Activity of Intact Mammalian Cells. J Membrane Biol 204, 49–56 (2005). https://doi.org/10.1007/s00232-005-0746-7
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/s00232-005-0746-7