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In the present experiments we expressed the rat kidney Na+-HCO3- cotransporter (rkNBC) in Xenopus laevis oocytes to reinvestigate the flux coupling ratio under improved measuring conditions. Essentially the current/voltage (I/V) relationship of isolated inside-out giant membrane patches was measured and the stoichiometric ratio was calculated from the reversal potential (VI=0) of the cotransport current (INBC). INBC was defined as that part of the total current that was suppressed when rkNBC was inhibited. Previously we have used the disulfonic stilbene DIDS to inhibit rkNBC, but we now found that tenidap or benzamil are better suited as inhibitors. Tenidap blocked rkNBC rapidly and reversibly both from the intra- and extracellular surface with half maximal inhibition at 13 micromol/l and it did not cause the same potentially disturbing side effects as DIDS. In addition, we found that the endogenous depolarization-induced Na+ conductance of the oocyte, which may compromise the I/V analysis, can be suppressed by applying 1 mmol/l amiloride to the cytosolic surface of the patch. The new measuring conditions greatly increased the yield of successful experiments. The distribution of 27 measurements of VI=0 obtained at near physiological Na+ and HCO3- concentrations and in absence of Cl-, K+ and cytosolic Ca2+ showed that the calculated stoichiometric ratios closely approached the value of 2 HCO3-:1 Na+ if the expression density of rkNBC was high. This result fully confirms our previous observations. Further experiments showed that the difference between the stoichiometric ratio of 3:1 observed in rat proximal tubule in vivo and the present value is not due to the temperature difference. We conclude that, depending on local modulatory influences, rkNBC can operate with different stoichiometric ratios and the present data and those reported in an accompanying publication [Müller-Berger et al., Pflügers Arch (2001) DOI 10.1007/s004240100592] show that these ratios are integer numbers i.e. either 2:1 or 3:1.

The Na+-HCO3 – cotransporter of rat kidney (rkNBC) was expressed in Xenopus laevis oocytes to test whether cytosolic Ca2+ ([Ca2+]i) affects the cotransport stoichiometry. The current/voltage relationship of giant inside-out membrane patches of rkNBC-expressing oocytes was measured at near-physiological Na+ and HCO3 – concentrations and the cotransport current, I NBC, was defined as the current inhibited by 0.25 mmol/l tenidap. Essentially, we determined the reversal potential (V I =0) of I NBC and the slope conductance (g NBC). The coupling ratio of HCO3 – to Na+ (q) was calculated from V I =0. As reported in the preceding publication [Ducoudret et al., Pflugers Arch (2001) DOI 10.1007/s004240100594], in Ca2+-free solutions q was 2:1. This did not change when [Ca2+]i was increased to 0.1 µmol/l. At 0.5 µmol/l, however, only a few patches showed q=2:1, while most patches exhibited q=3:1. This indicates that [Ca2+]i affected the transport function of membrane-resident rkNBC molecules, and the bimodal distribution of V I =0 points to an indirect effect possibly mediated by differently expressed Ca2+-dependent protein kinases. The shift in q was associated with the predicted near twofold increase in g NBC and was confirmed by measurements of V I =0 at different Na+ and HCO3 – concentrations. Because we previously observed that the cotransport in proximal tubule cells is susceptible to carbonic anhydrase (CA) inhibition, but only if it works at q=3:1, we propose that kNBC has three transport sites: when working at q=2:1 it binds 2 HCO3 –+1 Na+, and while at q=3:1 it binds 1 CO3 2–+1 HCO3 – +1 Na+. The latter is equivalent to the transfer of 3 HCO3 – +1 Na+, because in the presence of CA the generation of 1 CO3 2– on one side of the membrane and its disintegration on the other transiently liberates 1 CO2, which follows by diffusion. This model explains the increase in HCO3 – transport that is associated with the change in q from 2:1 to 3:1 by a selectivity change of a binding site from HCO3 – to CO3 2–. This is more likely than the induction of a new transport pouch for a third HCO3 – ion, which would require exceedingly large conformational changes of the transport protein.