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Phosphorylation‐Induced Modulation of pNBC1 Function: Distinct Roles for the Amino‐ and Carboxy‐Termini
Author(s) -
Gross E.,
Fedotoff O.,
Pushkin A.,
Abuladze N.,
Newman D.,
Kurtz I.
Publication year - 2003
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2003.042226
Subject(s) - phosphorylation , cotransporter , chemistry , stoichiometry , biochemistry , intracellular , biophysics , microbiology and biotechnology , sodium , biology , organic chemistry
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 HCO 3 − :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 Ser 982 . pNBC1 has the identical carboxy‐terminal consensus phosphorylation PKA site (KKGS 1026 ), and an additional site in its amino‐terminus (KRKT 49 ). 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 Ser 1026 following 8‐Br‐cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser 1026 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 Thr 49 , 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 HCO 3 − :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 Ser 1026 mediates the cAMP‐dependent shift in the stoichiometry of pNBC1, whereas Thr 49 plays an essential role in the cAMP‐induced increase in G DS .