The renal Na-HCO sub(3)-cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca super(2+) concentration

The Na super(+)-HCO sub(3) super(-)cotransporter of rat kidney (rkNBC) was expressed in Xenopus laevis oocytes to test whether cytosolic Ca super(2+) ([Ca super(2+)] sub(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 super(+) and HCO sub(3) super(-) concentrations and the cotransport current, I sub(NBC), was defined as the current inhibited by 0.25 mmol/l tenidap. Essentially, we determined the reversal potential (V sub(I=0)) of I sub(NBC) and the slope conductance (g sub(NBC)). The coupling ratio of HCO sub(3) super(-) to Na super(+) (q) was calculated from V sub(I=0). As reported in the preceding publication, in Ca super(2+)-free solutions q was 2:1. This did not change when [Ca super(2+)] sub(i) was increased to 0.1 mu mol/l. At 0.5 mu mol/l, however, only a few patches showed q=2:1, while most patches exhibited q=3:1. This indicates that [Ca super(2+)] sub(i) affected the transport function of membrane-resident rkNBC molecules, and the bimodal distribution of V sub(I=0) points to an indirect effect possibly mediated by differently expressed Ca super(2+)-dependent protein kinases. The shift in q was associated with the predicted near twofold increase in g sub(NBC) and was confirmed by measurements of V sub(I=0) at different Na super(+) and HCO sub(3) super(-) 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:l, we propose that kNBC has three transport sites: when working at q=2:1 it binds 2 HCO sub(3) super(-)+1 Na super(+), and while at q=3:1 it binds 1 CO sub(3) super(2-)+1 HCO sub(3) super(-)+1 Na super(+). The latter is equivalent to the transfer of 3 HCO sub(3) super(-)+1 Na super(+), because in the presence of CA the generation of 1 CO sub(3) super(2-) on one side of the membrane and its disintegration on the other transiently liberates 1 CO sub(2), which follows by diffusion. This model explains the increase in HCO sub(3) super(-) transport that is associated with the change in q from 2:1 to 3:1 by a selectivity change of a binding site from HCO sub(3) super(-) to CO sub(3) super(2-). This is more likely than the induction of a new transport pouch for a third HCO sub(3) super(-) ion, which would require exceedingly large conformational changes of the transport protein.