Enzymatic Activity of Renal H-K-ATPase in the Outer Medullary Collecting Duct of Transgenic Mice


The H-K-ATPase (HKA), a potassium-dependent proton transporter in the outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis. The OMCD contains two HKA isoforms; gastric (HKAα1), dominant under normal dietary conditions (ND), and colonic (HKAα2), induced under a K-free diet (KD). The enzymatic activity (EA) of HKA in the OMCD is incompletely understood. The focus of the present study is elucidating the EA of the HKA in HKAα1 and HKAα2 knockout (KO) mice under ND and KD. KO mice were subjected to ND or KD for 10 days. Ten OMCD tubules were extracted, half placed in potassium-free media (Solution 2), half in potassium-containing media (Solution 3). Fluorescence measurements are based on the hydrolysis of ATP to ADP, coupled with the oxidation of NADH. ADP is determined by a decrease in NADH fluorescence. In K presence, NADH fluorescence of HKAα1 KO mice read 13.5 ± 0.7 ppm for ND and 10.3 ± 0.2 ppm for KD, indicating stimulation of the colonic isoform. HKAα2 KO mice averaged 6.8 ± 0.3 ppm for ND and 5.4 ± 0.3 ppm for KD in solution 2 (p < 0.002). Solution 3 readings were 6.0 ± 0.3 ppm for ND and 4.6 ± 0.2 ppm for KD (p < 0.0005). K addition produced significant changes in NADH fluorescence of ND and KD KO mice. The results demonstrated potassium depletion’s association with increased EA of H-K-ATPase in OMCD, consistent with the activation of HKAα2 isoform. A significant difference in ATP production in HKAα2 KO mice is likely due to enhanced EA of H-ATPase under potassium depletion.

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Downing, T. , Mangla, A. , Banta, M. and Nakamura, S. (2014) Enzymatic Activity of Renal H-K-ATPase in the Outer Medullary Collecting Duct of Transgenic Mice. International Journal of Clinical Medicine, 5, 1239-1247. doi: 10.4236/ijcm.2014.520158.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Nakamura, S., Amlal, H., Schultheis, P., Galla, J., Shull, G. and Soleimani, M. (1999) HCO3-Reabsorption in Renal Collecting Duct of NHE-3 Deficient Mouse: A Compensatory Response. American Journal of Physiology, 276, 914-921.
[2] Nakamura, S., Wang, Z., Galla, J. and Soleimani, M. (1998) K+ Depletion Increases HCO3-Reabsorption in OMCD by Activation of Colonic H-K-ATPase. American Journal of Physiology, 274, 687-692.
[3] Nakamura, S., Amlal, H., Soleimani, M. and Galla, J. (2000) Pathways for HCO3-Reabsorption in Mouse Medullary Collecting Duct Segments. Journal of Laboratory and Clinical Medicine, 136, 218-223.
[4] Garg, L. and Narang, N. (1988) Oubain-Insensitive K-Adenosine Triphosphatase in Distal Nephron Segments of the Rabbit. The Journal of Clinical Investigation, 81, 1204-1208.
[5] Nakamura, S., Amlal, H., Soleimani, M. and Galla, J. (1999) Secretion in Inner Medullary Collecting Duct in Potassium Deprivation: Role of Colonic H+-K+-ATPase. Kidney International, 56, 2160-2167.
[6] Garg, L., Knepper, M. and Burg, M. (1981) Mineralocorticoid Effects of Na-K-ATPase in Individual Nephron Segments. American Journal of Physiology, 240, 536-544.
[7] Zemkova, H., Teisinger, J. and Vyskocil, F. (1988) Inhinition of the Electrogenic Na,K Pump and Na,K-ATPase Activity by Tetraethlyammonium, Tetrabutylammonium, and Apamin. Journal of Neuroscience Research, 19, 497-503.
[8] Nunez, R., Calva, E. and Marsh, M. (1975) Nicotinamide Adenine Dinucleotide Degradation in Infarcted Cardiac Muscle. Recent Advances in Studies on Cardiac Structure and Metabolism, 10, 241-250.
[9] Wilder, M., Do, T., Atmomarsono, M., Tran, T., Truong, Q. and Yang, W. (2000) Characterization of Na/K-ATPase in Macrobrachiumrosenbergii and the Effects of Changing Salinity on Enzymatic Activity. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 123, 377-388.
[10] Meneton, P., Schultheis, P. and Greeb, J. (1998) Increased Sensitivity to K+ Deprivation in Colonic H,K-ATPase Deficient Mice. Journal of Clinical Investigation, 101, 536-542.
[11] Spicer, Z., Miller, M., Andringa, A., Riddle, T., Duffy, J., Doetschman, T. and Shull, G. (2000) Stomachs of Mice Lacking the Gastric H,K-ATPase Alpha-Subunit Have Ach-lorhydria, Abnormal Parietal Cells and Ciliated Metaphasia. The Journal of Biological Chemistry, 275, 21555-21565.
[12] Nakamura, S. (2006) H+-ATPase Activity in Selective Disruption of H+-K+-ATPase Alpha 1 Gene of Mice under Normal and K-Depleted Conditions. The Journal of Laboratory and Clinical Medicine, 147, 45-51.
[13] Guojun, W., Ravelletee, J. and Nakamura, S. (2013) Potassium Depletion Stimulates Beta Subunit of Colonic H+-K+-ATPase in Mice. International Journal of Clinical Medicine, 4, 244-250.
[14] Xiaming, Z., Nakamura, S., Xia, S. and Wingo, C. (2001) Increase CO2 Stimulates K/Rb Reabsorption Mediated by H-K-ATPase in CCD of Potassium-Restricted Rabbit. American Journal of Physiology, 281, 366-373.
[15] Swarts, H., Koenderink, J., Willems, P. and De Pont, J. (2007) The Human Non-Gastric H,K-ATPase Has a Different Cation Specificity than the Rat Enzyme. Biochimica et Biophysica Acta, 1768, 580-589.
[16] Swarts, H., Koenderink, J., Willems, P. and De Pont, J. (2005) The Non-Gastric H,K-ATPase Is Oligomycin-Sensitive and Can Function as an H+, -ATPase. Journal of Biological Chemistry, 280, 33115-33122.
[17] Streif, D., Iglseder, E., Hauser-Kronberger, C., Fink, K., Jakab, M. and Ritter, M. (2011) Expression of the Non-Gastric H+/K+ ATPase ATP12A in Normal and Pathological Human Prostate Tissue. Cellular Physiology and Biochemistry, 28, 1287-1294.
[18] Novak, I., Wang, J., Henriksen, K., Haanes, K., Krabbe, S., Nitschke, R. and Hede, S. (2011) Pancreatic Bicarbonate Secretion Involves Two Proton Pumps. The Journal of Biological Chemistry, 286, 288-289.
[19] Fischer, H. and Widdicombe, J. (2006) Mechanisms of Acid and Base Secretion by the Airway Epithelium. Journal of Membrane Biology, 211, 139-150.
[20] Lerner, M., Lemke, D., Bertram, H., Schillers, H., Oberleithner, H., Caplan, M. and Reinhardt, J. (2006) An Extracellular Loop of the Human Non-Gastric H,K-ATPase Alpha-Subunit Is Involved in Apical Plasma Membrane Polarization. Cellular Physiology and Biochemistry, 18, 75-84.
[21] Qiu, L., Swarts, H., Tonk, E., Willems, P., Koenderink, J. and De Pont, J. (2006) Conversion of the Low Affinity Ouabain-Binding Site of Non-Gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids. The Journal of Biological Chemistry, 281, 13533-13539.
[22] Crambert, G., Horisberger, J.D., Modyanov, N.N. and Geering, K. (2002) Human Nongastric H+-K+-ATPase: Transport Properties of ATP1al1 Assembled with Different Beta Subunits. American Journal of Physiology—Cell Physiology, 283, 305-314.
[23] Swarts, H., Koenderink, J., Willems, P. and De Pont, J. (2009) The Non-Gastric H,K-ATPase as a Tool to Study the Ouabain-Binding Site in Na,K-ATPase. Pflügers Arc-hiv—European Journal of Physiology, 457, 623-634.
[24] Radkov, R., Kharoubi-Hess, S., Schaer, D., Modyanov, N., Geering, K. and Horisberger, J. (2007) Role of Homologous ASP334 and GLU319 in Human Non-Gastric H,K- and Na,K-ATPases in Cardiac Glycoside Binding. Biochemical and Biophysical Research Communications, 356, 142-146.
[25] Pestov, N.B., Korneenko, T.V., Radkov, R., Zhao, H., Shakhparonov, M.I. and Modyanov, N.N. (2004) Identification of the Beta-Subunit for Nongastric H-K-ATPase in Rat Anterior Prostate. American Journal of Physiology—Cell Physiology, 286, C1229-C1237.
[26] Codina, J., Delmas-Mata, J. and Dubose, T. (1998) The Alpha-Subunit of the Colonic H+,K+-ATPase Assembles with Beta1-Na+, K+-ATPase in Kidney and Distal Colon. The Journal of Biological Chemistry, 273, 7894-7899.
[27] Codina, J., Pressley, T. and Dubose, T. (1997) Effect of Chronic Hypokalemia on H+-K+-ATPase Expression in Rat Colon. American Journal of Physiology, 272, 22-30.
[28] Jaisser, F., Escoubet, B., Coutry, N., Eugene, E., Bonvalet, J. and Farman, N. (1996) Differential Regulation of Putative K+-ATPase by Low-K+ Diet and Corticosteroids in Rat Distal Colon and Kidney. American Journal of Physiology, 270, 679-687.
[29] Sangan, P., Rajendran, V., Mann, A., Kashgarian, M. and Binder, H. (1997) Regulation of Colonic H-K-ATPase in Large Intestine and Kidney by Dietary Na Depletion and Dietary K Depletion. American Journal of Physiology, 272, 685-696.
[30] Burnay, M., Crambert, G., Kharoubi-Hess, S., Geering, K. and Horisberger, J. (2003) Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment. The Journal of Biological Chemistry, 278, 19237-19244.
[31] Jaisser, F. (1996) Molecular and Functional Diversity of Na,K-ATPase and Renal H,K-ATPases. Nephrologie, 17, 401-408.
[32] Nakamura, S., Amlal, H., Galla, J. and So-leimani, M. (1998) Colonic H+-K+-ATPase Is Induced and Mediates Increased Reabsorption in Inner Medullary Collecing Duct in Potassium Depletion. Kidney International, 54, 1233-1239.
[33] Zhao, N., Sui, G., Li, N., Wu, X., Yang, Z., Lai, G., Yang, Y., Lin, Z. and Tang, X. (2011) Study on Activities and Protein and Gene Expression of Renal H+-K+-ATPase in Rats Subchronic Exposed to Trimethyltin Chloride. Chinese Journal of Industrial Hygiene and Occupational Diseases, 29, 299-302.
[34] Poulsen, H., Morth, P., Egebjerg, J. and Nissen, P. (2010) Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase. FEBS Letters, 584, 2589-2595.
[35] Witzke, A., Lindner, K., Munson, K. and Apell, H. (2010) Inhibition of the Gastric H,K-ATPase by Clotrimazole. Biochemistry, 49, 4524-4532.

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