Phosphorylation of Ser(982) in the sodium bicarbonate cotransporter kNBC1 shifts the HCO(3)(-) : Na(+) stoichiometry from 3 : 1 to 2 : 1 in murine proximal tubule cells

Abstract

1. Adenosine 3',5'-cyclic monophosphate (cAMP) modulates proximal tubule sodium and bicarbonate absorption by decreasing the rate of apical Na(+)-H(+) exchange and basolateral sodium bicarbonate efflux, through activation of protein kinase A (PKA). The electrogenic sodium bicarbonate cotransporter kNBC1 mediates basolateral sodium and bicarbonate efflux in the proximal tubule by coupling the transport of 1 Na(+) cation to that of 3 HCO(3)(-) anions. In this work we studied the effects of cAMP on the function of kNBC1 expressed heterologously in a proximal tubule cell line. 2. A mouse renal proximal tubule cell line, deficient in electrogenic sodium bicarbonate cotransport function, was transfected with kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The HCO(3)(-) : Na(+) stoichiometry of kNBC1 was calculated from its reversal potential by measuring the current-voltage relationships of the cotransporter at different Na(+) concentration gradients. 3. Addition of the potent cAMP agonist 8-Br-cAMP caused the stoichiometry of kNBC1 to shift from 3 HCO(3)(-) : 1 Na(+) to 2 HCO(3)(-) : 1 Na(+). Pretreatment of the cells with the PKA inhibitor H-89 abolished the effect of the agonist on the stoichiometry change. Replacing Ser(982) at the C-terminus consensus PKA phosphorylation site with alanine resulted in a failure of PKA to phosphorylate the transporter and induce a stoichiometry shift. 4. Our data indicate that cAMP modulates the stoichiometry of kNBC1 through activation of PKA. The change in stoichiometry from 3 : 1 to 2 : 1 is predicted to cause a shift in the direction of basolateral membrane sodium bicarbonate transport from efflux to influx. Ser(982) in the C-terminus of kNBC1 is a target for PKA phosphorylation. This is the first example of modulation of the stoichiometry of a membrane transporter by phosphorylation.

Figure 1. Current-voltage relationships of kNBC1 constructs

A, current-voltage relationships of WT kNBC1 (open symbols) and EGFP-WT-kNBC1 (filled symbols) in the absence (squares) and presence (triangles) of 8-Br-cAMP. B, effect of 8-Br-cAMP on I–V relationships of WT kNBC1 (open symbols) and EGFP-WT-kNBC1 (filled symbols) in cells pre-incubated with H-85 (triangles) or H-89 (squares). C, I–V relationships of S982A-kNBC1 (open symbols) and EGFP-S982A-kNBC1 (filled symbols) in the absence (squares) and presence (triangles) of 8-Br-cAMP. D, effect of 8-Br-cAMP on I–V relationships of S982A-kNBC1 (open symbols) and EGFP-WT-kNBC1 (filled symbols) in cells pre-incubated with H-85 (triangles) or H-89 (squares). I–V relationships of mock-transfected cells are shown with circles in A–D. All I–V relationships were measured at 5-fold Na⁺ concentration gradient (apical/basolateral = 10 mm/50 mm) in transformed mPCT cells.

Figure 2. Membrane localization of EGFP-WT-kNBC1

A, cells transfected with the EGFP-WT-kNBC1 construct showing plasma membrane localization of the fusion protein. B, mock-transfected cells.

Figure 3. Phosphorylation of kNBC1 by PKA

A, in vitro phosphorylation of WT kNBC1 (lanes 1 and 2) and the S982A-kNBC1 mutant (lanes 3 and 4) isolated from untreated mPCT cells (lanes 1 and 3) or cells treated with 100 μm 8-Br-cAMP (lanes 2 and 4) for 15 min. Immunoprecipitated EGFP-WT-kNBC1 and the EGFP-S982A-kNBC1 mutant were eluted from protein A-Sepharose beads and then phosphorylated in vitro using the PKA catalytic subunit and [γ-³²P]ATP. A representative experiment is shown. B, the amount of the wild-type and mutant kNBC1 proteins used in these in vitro phosphorylation experiments was quantified using Western blotting with an affinity purified rabbit polyclonal kNBC1 antibody (Bok et al. 2001). Treatment of the cells with 8-Br-cAMP did not affect the size of EGFP-kNBC1 and the EGFP-S982A-kNBC1 mutant.