The renal Na-HCO3-cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca2+ concentration

The Na+-HCO3- cotransporter of rat kidney (rkNBC) was expressed in Xenopus laevis oocytes to test whether cytosolic Ca2+ ([Ca2+](i)) affects the cotra nsport stoichiometry. The current/voltage relationship of giant inside-out membrane patches of rkNBC-expressing oocytes was measured at near-physiolog ical Na+ and HCO3- concentrations and the cotransport current, I-NBC, was d efined as the current inhibited by 0.25 mmol/l tenidap. Essentially, we det ermined 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 VI-0. A s reported in the preceding publication [Ducoudret et al., Pflugers Arch (2 001) DOI 10.1007/s004240100594], in Ca2+-free solutions q was 2:1. This did not change when [Ca2+](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 [Ca2+](i) affected the transport function of membran e-resident rkNBC molecules, and the bimodal distribution of V-I=0 points to an indirect effect possibly mediated by differently expressed Ca2+-depende nt protein kinases. The shift in q was associated with the predicted near t wofold increase in g(NBC) and was confirmed by measurements of V-I=0 at dif ferent Na+ and HCO3- concentrations. Because we previously observed that th e 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 t hree transport sites: when working at q=2:1 it binds 2 HCO3-+1 Na+, and whi le at q=3:1 it binds 1 CO32-+1 HCO3- +1 Na+. The latter is equivalent to th e transfer of 3 HCO3- +1 Na+, because in the presence of CA the generation of 1 CO32- on one side of the membrane and its disintegration on the other transiently liberates I CO2 which follows by diffusion. This model explains the increase in HCO3- transport that is associated with the change in q fr om 2:1 to 3:1 by a selectivity change of a binding site from HCO3- to CO32- . This is more likely than the induction of a new transport pouch for a thi rd HCO3- ion, which would require exceedingly large conformational changes of the transport protein.