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Pflügers Archiv

, Volume 438, Issue 3, pp 322–329 | Cite as

Stoichiometry of the rat kidney Na+-HCO3 cotransporter expressed in Xenopus laevis oocytes

  • M. Heyer
  • S. Müller-Berger
  • M. F. Romero
  • W. F. Boron
  • E. Frömter
Original Article

Abstract 

The rat kidney Na+-HCO3 cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3 as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4,4’-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3 to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 µmol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3 concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3 concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.

Key words ATP DIDS Intracellular and extracellular Na+ Renal Na+-HCO3 cotransporter Transport stoichiometry Xenopus laevis oocytes 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1999

Authors and Affiliations

  • M. Heyer
    • 1
  • S. Müller-Berger
    • 1
  • M. F. Romero
    • 2
  • W. F. Boron
    • 3
  • E. Frömter
    • 1
  1. 1.Zentrum der Physiologie, Klinikum der JW Goethe Universität, D-60590 Frankfurt/Main, GermanyDE
  2. 2.Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106–4970, USAUS
  3. 3.Department of Cell and Molecular Physiology, Yale University, School of Medicine, New Haven CT 06510–3219 USAUS

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