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

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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 (I(NBC)) 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 I(NBC) 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., Pflugers 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.

Original languageEnglish (US)
Pages (from-to)322-329
Number of pages8
JournalPflugers Archiv European Journal of Physiology
Volume438
Issue number3
DOIs
StatePublished - 1999

Keywords

  • ATP
  • DIDS
  • Intracellular and extracellular Na
  • Renal Na-HCO cotransporter
  • Transport stoichiometry
  • Xenopus laevis oocytes

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

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