Abstract
BackgroundThe renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. ResultsNa+- and HCO3--dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10-5 M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10-4 M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. ConclusionRegulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.
Highlights
The renal and intestinal electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues
The longer N-terminal tail of pNBC1 contains unique phosphorylation sites for PKA (Thr49), PKC (Ser38 and Ser65), and casein kinase II (Ser68), which are not found in the kNBC1 sequence and of which at least the cAMP-dependent site is relevant for transporter regulation [7,9]
To determine the distribution of NBC1 subtypes in HEK293 cells compared to native tissue, we first performed Polymerase chain reaction (PCR) analysis (Figure 1)
Summary
The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. BMC Cell Biology 2008, 9:70 http://www.biomedcentral.com/1471-2121/9/70 the intracellular supply of HCO3- destined for secretion [2]. These striking differences in function and transport direction have prompted studies to elucidate the structural and regulatory properties of the respective transporters. The renal (kNBC1) and intestinal (pNBC1) NBC subtypes possess a common C-terminal PKA-dependent phosphorylation site (Ser982 and Ser1026, respectively), which was reported to determine transport stoichiometry in renal cells [7,8]. The longer N-terminal tail of pNBC1 contains unique phosphorylation sites for PKA (Thr49), PKC (Ser and Ser65), and casein kinase II (Ser68), which are not found in the kNBC1 sequence and of which at least the cAMP-dependent site is relevant for transporter regulation [7,9]
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