| Abstrakt: |
Astrocytes are pivotal responders to alterations of extracellular pH, primarily by regulation of their principal acid‐base transporter, the membrane‐bound electrogenic Na+/bicarbonate cotransporter 1 (NBCe1). Here, we describe amammalian target of rapamycin (mTOR)‐dependent and NBCe1‐mediated astroglial response to extracellular acidosis. Using primary mouse cortical astrocytes, we investigated the effect of long‐term extracellular metabolic acidosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, and phosphoproteomic analysis. The results showed significant increase of NBCe1‐mediated recovery of intracellular pH from acidification in WT astrocytes, but not in cortical astrocytes from NBCe1‐deficient mice. Acidosis‐induced upregulation of NBCe1 activity was prevented following inhibition of mTOR signaling by rapamycin. Yet, during acidosis or following exposure of astrocytes to rapamycin, surface protein abundance of NBCe1 remained ‐unchanged. Mutational analysis in HeLa cells suggested that NBCe1 activity was dependent on phosphorylation state of Ser245, a residue conserved in all NBCe1 variants. Moreover, phosphorylation state of Ser245 is regulated by mTOR and is inversely correlated with NBCe1 transport activity. Our results identify pSer245 as a novel regulator of NBCe1 functional expression. We propose that context‐dependent and mTOR‐mediated multisite phosphorylation of serine residues of NBCe1 is likely to be a potent mechanism contributing to the response of astrocytes to acid/base challenges during pathophysiological conditions. [ABSTRACT FROM AUTHOR] |
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