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  • Title: Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.
    Author: Heyer M, Müller-Berger S, Romero MF, Boron WF, Frömter E.
    Journal: Pflugers Arch; 1999 Aug; 438(3):322-9. PubMed ID: 10398862.
    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 micromol/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.
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