Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.
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.
Zentrum der Physiologie, Klinikum der JW Goethe Universität, D-60590 Frankfurt/Main, Germany.
M
M
Heyer
S
S
Müller-Berger
M F
MF
Romero
W F
WF
Boron
E
E
Frömter
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D015870:Gene Expression; D007668:Kidney / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters; D014982:Xenopus laevis
D001639:Bicarbonates; D002352:Carrier Proteins; D011994:Recombinant Proteins; D027982:Sodium-Bicarbonate Symporters; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid