PIP2 hydrolysis stimulates the electrogenic Na+-bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes.
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., Pflügers 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.