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.
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.
Gross
E
E
Department of Urology, Case Western Reserve University and VA Medical Center, Cleveland, OH 44106, USA. ezg@po.cwru.edu
Hawkins
K
K
Pushkin
A
A
Sassani
P
P
Dukkipati
R
R
Abuladze
N
N
Hopfer
U
U
Kurtz
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002460:Cell Line; D055598:Chemical Phenomena; D002621:Chemistry; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000819:agonists / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008433:Mathematics; D051379:Mice; D010766:Phosphorylation; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*
D001639:Bicarbonates; D027982:Sodium-Bicarbonate Symporters; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases