Revisiting the role of Ser982 phosphorylation in stoichiometry shift of the electrogenic Na⁺ /qHCO₃⁻ cotransporter NBCe1

In most cell types and heterologous expression systems, the electrogenic sodium‐bicar-bonate cotransporter NBCe1 operates with a 1Na⁺–2HCO3⁻ stoichiometry that, given typical trans-membrane electrochemical gradients, promotes Na+ and HCO3⁻ influx. However, NBCe1 in the kidney mediates HCO3⁻ efflux (HCO3⁻ reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy‐terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non‐phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phospho-proteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides in-cluding phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characteri-zation, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3⁻ efflux) than in murine colons (in which NBCe1 mediates HCO3⁻ influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoi-chiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.