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Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini

E. Gross *†, O. Fedotoff *‡, A. Pushkin ‡, N. Abuladze ‡, D. Newman ‡ and I. Kurtz ‡

The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO₃^-:Na⁺ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser⁹⁸². pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS¹⁰²⁶), and an additional site in its amino-terminus (KRKT⁴⁹). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser¹⁰²⁶ following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser¹⁰²⁶ with Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (G_DS) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr⁴⁹, which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in G_DS. In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO₃^-:Na⁺ stoichiometry of 2:1, 8-Br-cAMP increased G_DS by ~90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser¹⁰²⁶ mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr⁴⁹ plays an essential role in the cAMP-induced increase in G_DS.

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