The renal Na-HCO3-cotransporter expressed in Xenopus laevis oocytes:: inhibition by tenidap and benzamil and effect of temperature on transport rate and stoichiometry

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 (V-l=0) of the cotransport current (I-NBC). I-NBC 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 mu mol/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 V-l=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 [Muller-Berger et al., Pflugers Arch (2001) DOI 10.1007/s004240100592] show that these ratios are integer numbers i.e. either 2:1 or 3: 1.