Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3- Cotransporter NBCe1.

Thamer A Alsufayan, Evan J Myers, Bianca N Quade, Clayton T Brady, Aniko Marshall, Nayem Haque, Michael E Duffey, Mark D Parker

International journal of molecular sciences 2021 Nov 26

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In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+-2HCO3- stoichiometry that, given typical transmembrane 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, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including 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, characterization, 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 stoichiometry 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.

Citation

Thamer A Alsufayan, Evan J Myers, Bianca N Quade, Clayton T Brady, Aniko Marshall, Nayem Haque, Michael E Duffey, Mark D Parker. Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3- Cotransporter NBCe1. International journal of molecular sciences. 2021 Nov 26;22(23)