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Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp⁹⁸⁶, Asp⁹⁸⁸ and kNBC1-carbonic anhydrase II binding

Eitan Gross *†‡, Alexander Pushkin§, Natalia Abuladze§, Olga Fedotoff ‡§ and Ira Kurtz§

The HCO₃^- : Na⁺ cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (E_rev) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1-Ser⁹⁸² in the carboxy-terminus of kNBC1 (kNBC1-Ct), by cAMP-protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1-Ser⁹⁸², or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate-binding site. In the current study, we examined the role of the negative charge on Ser⁹⁸²-phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)-tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1-Ser⁹⁸². The data indicate that both kNBC1-Asp⁹⁸⁶ and kNBC1-Asp⁹⁸⁸, but not kNBC1-Asp⁹⁸⁹, are required for the phosphorylation-induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy-terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1-Ct with a K_D of 160 ± 10 nM. Acetazolamide inhibited the short-circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8-Br-cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1-Ser⁹⁸² is unphosphorylated.

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