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. 1999 Jun;76(6):3066–3075. doi: 10.1016/S0006-3495(99)77459-X

Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule.

E Gross 1, U Hopfer 1
PMCID: PMC1300276  PMID: 10354432

Abstract

The effects of pH on cotransporter kinetics were studied in renal proximal tubule cells. Cells were grown to confluence on permeable support, mounted in an Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, dinitrostilbene-disulfonate-sensitive current (DeltaI) was Na+ and HCO3- dependent and therefore was taken as flux through the cotransporter. When the pH of the perfusing solution was changed between 6.0 and 8.0, the conductance attributable to the cotransporter showed a maximum between pH 7.25 and pH 7.50. A similar profile was observed in the presence of a pH gradient when the pH of the apical solutions was varied between 7.0 and 8.0 (basal pH lower by 1), but not when the pH of the basal solution was varied between 7.0 and 8.0 (apical pH lower by 1 unit). To delineate the kinetic basis for these observations, DeltaI-voltage curves were obtained as a function of Na+ and HCO3- concentrations and analyzed on the basis of a kinetic cotransporter model. Increases in pH from 7.0 to 8.0 decreased the binding constants for the intracellular and extracellular substrates by a factor of 2. Furthermore, the electrical parameters that describe the interaction strength between the electric field and substrate binding or charge on the unloaded transporter increased by four- to fivefold. These data can be explained by a channel-like structure of the cotransporter, whose configuration is modified by intracellular pH such that, with increasing pH, binding of substrate to the carrier is sterically hindered but electrically facilitated.

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Selected References

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  1. Acevedo M. Effect of acetyl choline on ion transport in sheep tracheal epithelium. Pflugers Arch. 1994 Jul;427(5-6):543–546. doi: 10.1007/BF00374272. [DOI] [PubMed] [Google Scholar]
  2. Alpern R. J., Chambers M. Cell pH in the rat proximal convoluted tubule. Regulation by luminal and peritubular pH and sodium concentration. J Clin Invest. 1986 Aug;78(2):502–510. doi: 10.1172/JCI112602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alpern R. J. Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. J Gen Physiol. 1985 Nov;86(5):613–636. doi: 10.1085/jgp.86.5.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aronson P. S. Mechanisms of active H+ secretion in the proximal tubule. Am J Physiol. 1983 Dec;245(6):F647–F659. doi: 10.1152/ajprenal.1983.245.6.F647. [DOI] [PubMed] [Google Scholar]
  5. Aronson P. S., Nee J., Suhm M. A. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature. 1982 Sep 9;299(5879):161–163. doi: 10.1038/299161a0. [DOI] [PubMed] [Google Scholar]
  6. Backman K., Harrison B., Meysenberg M., Schwartz C., Germann W. Inactivation of a volume-sensitive basolateral potassium conductance in turtle colon: effect of metabolic inhibitors. Biochim Biophys Acta. 1992 Mar 23;1105(1):89–96. doi: 10.1016/0005-2736(92)90166-j. [DOI] [PubMed] [Google Scholar]
  7. Biagi B. A., Sohtell M. Electrophysiology of basolateral bicarbonate transport in the rabbit proximal tubule. Am J Physiol. 1986 Feb;250(2 Pt 2):F267–F272. doi: 10.1152/ajprenal.1986.250.2.F267. [DOI] [PubMed] [Google Scholar]
  8. Boorer K. J., Frommer W. B., Bush D. R., Kreman M., Loo D. D., Wright E. M. Kinetics and specificity of a H+/amino acid transporter from Arabidopsis thaliana. J Biol Chem. 1996 Jan 26;271(4):2213–2220. doi: 10.1074/jbc.271.4.2213. [DOI] [PubMed] [Google Scholar]
  9. Boron W. F., Boulpaep E. L. Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport. J Gen Physiol. 1983 Jan;81(1):53–94. doi: 10.1085/jgp.81.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boron W. F., Boulpaep E. L. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol. 1983 Jan;81(1):29–52. doi: 10.1085/jgp.81.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Boron W. F., Knakal R. C. Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-. J Gen Physiol. 1989 Jan;93(1):123–150. doi: 10.1085/jgp.93.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chao J., Graves D. J. pH dependence of the kinetic parameters of maltodextrin phosphorylase. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1398–1403. doi: 10.1016/0006-291x(70)90022-7. [DOI] [PubMed] [Google Scholar]
  13. Coppola S., Frömter E. An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules. II. Effect of micromolar concentrations. Pflugers Arch. 1994 May;427(1-2):151–156. doi: 10.1007/BF00585954. [DOI] [PubMed] [Google Scholar]
  14. Eskandari S., Loo D. D., Dai G., Levy O., Wright E. M., Carrasco N. Thyroid Na+/I- symporter. Mechanism, stoichiometry, and specificity. J Biol Chem. 1997 Oct 24;272(43):27230–27238. doi: 10.1074/jbc.272.43.27230. [DOI] [PubMed] [Google Scholar]
  15. Forster I., Hernando N., Biber J., Murer H. The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2). J Gen Physiol. 1998 Jul;112(1):1–18. doi: 10.1085/jgp.112.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grinstein S., Cohen S., Rothstein A. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J Gen Physiol. 1984 Mar;83(3):341–369. doi: 10.1085/jgp.83.3.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gross E., Hopfer U. Activity and stoichiometry of Na+:HCO3- cotransport in immortalized renal proximal tubule cells. J Membr Biol. 1996 Aug;152(3):245–252. doi: 10.1007/s002329900102. [DOI] [PubMed] [Google Scholar]
  18. Gross E., Hopfer U. Voltage and cosubstrate dependence of the Na-HCO3 cotransporter kinetics in renal proximal tubule cells. Biophys J. 1998 Aug;75(2):810–824. doi: 10.1016/S0006-3495(98)77570-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hager K., Hazama A., Kwon H. M., Loo D. D., Handler J. S., Wright E. M. Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes. J Membr Biol. 1995 Jan;143(2):103–113. doi: 10.1007/BF00234656. [DOI] [PubMed] [Google Scholar]
  20. Illek B., Fischer H., Clauss W. Quinidine-sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine. J Comp Physiol B. 1993;163(7):556–562. doi: 10.1007/BF00302114. [DOI] [PubMed] [Google Scholar]
  21. Jordan P. C. How pore mouth charge distributions alter the permeability of transmembrane ionic channels. Biophys J. 1987 Feb;51(2):297–311. doi: 10.1016/S0006-3495(87)83336-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kirk K. L., Dawson D. C. Basolateral potassium channel in turtle colon. Evidence for single-file ion flow. J Gen Physiol. 1983 Sep;82(3):297–313. doi: 10.1085/jgp.82.3.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klamo E. M., Drew M. E., Landfear S. M., Kavanaugh M. P. Kinetics and stoichiometry of a proton/myo-inositol cotransporter. J Biol Chem. 1996 Jun 21;271(25):14937–14943. doi: 10.1074/jbc.271.25.14937. [DOI] [PubMed] [Google Scholar]
  24. Läuger P., Jauch P. Microscopic description of voltage effects on ion-driven cotransport systems. J Membr Biol. 1986;91(3):275–284. doi: 10.1007/BF01868820. [DOI] [PubMed] [Google Scholar]
  25. Mackenzie B., Loo D. D., Fei Y., Liu W. J., Ganapathy V., Leibach F. H., Wright E. M. Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1. J Biol Chem. 1996 Mar 8;271(10):5430–5437. doi: 10.1074/jbc.271.10.5430. [DOI] [PubMed] [Google Scholar]
  26. Mackenzie B., Loo D. D., Wright E. M. Relationships between Na+/glucose cotransporter (SGLT1) currents and fluxes. J Membr Biol. 1998 Mar 15;162(2):101–106. doi: 10.1007/s002329900347. [DOI] [PubMed] [Google Scholar]
  27. Ottolenghi P. The effects of hydrogen ion concentration on the simplest steady-state enzyme systems. Biochem J. 1971 Jul;123(3):445–453. doi: 10.1042/bj1230445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Panayotova-Heiermann M., Loo D. D., Wright E. M. Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter. J Biol Chem. 1995 Nov 10;270(45):27099–27105. doi: 10.1074/jbc.270.45.27099. [DOI] [PubMed] [Google Scholar]
  29. Parent L., Supplisson S., Loo D. D., Wright E. M. Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions. J Membr Biol. 1992 Jan;125(1):63–79. doi: 10.1007/BF00235798. [DOI] [PubMed] [Google Scholar]
  30. Soleimani M., Lesoine G. A., Bergman J. A., McKinney T. D. A pH modifier site regulates activity of the Na+:HCO3- cotransporter in basolateral membranes of kidney proximal tubules. J Clin Invest. 1991 Oct;88(4):1135–1140. doi: 10.1172/JCI115413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Woost P. G., Orosz D. E., Jin W., Frisa P. S., Jacobberger J. W., Douglas J. G., Hopfer U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int. 1996 Jul;50(1):125–134. doi: 10.1038/ki.1996.295. [DOI] [PubMed] [Google Scholar]
  32. Yoshitomi K., Frömter E. How big is the electrochemical potential difference of Na+ across rat renal proximal tubular cell membranes in vivo? Pflugers Arch. 1985;405 (Suppl 1):S121–S126. doi: 10.1007/BF00581792. [DOI] [PubMed] [Google Scholar]
  33. Yun C. H., Lamprecht G., Forster D. V., Sidor A. NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem. 1998 Oct 2;273(40):25856–25863. doi: 10.1074/jbc.273.40.25856. [DOI] [PubMed] [Google Scholar]

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