Regulation of electrogenic Na⁺/HCO₃⁻ cotransporter 1 (NBCe1) function and its dependence on m‐TOR mediated phosphorylation of Ser²⁴⁵

Abstract

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 NBCe1mediated 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 Ser 245 , a residue conserved in all NBCe1 variants. Moreover, phosphorylation state of Ser 245 is regulated by mTOR and is inversely correlated with NBCe1 transport activity. Our results identify pSer 245 as a novel regulator of NBCe1 functional expression. We propose that contextdependent 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.

Cell-Type Dependent Regulation of the Electrogenic Na+/HCO3− Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma

“…First, NBCe1 protein abundance was determined by immunoblot analysis. Using an antibody raised against NBCe1, strong immunoreactive bands at ~110 kDa and ~130 kDa were detected in cell homogenates of all GBM cellular states, as previously described for mouse hippocampal and cortical astrocytes [ 14 , 15 , 16 ], ( Figure 1 A). These results were further confirmed by immunofluorescence, as shown in Figure 1 B,D.…”

“…We have previously shown that post-translational modifications of NBCe1, such as phosphorylation at distinct serine residues is a potent regulatory mechanism for NBCe1 transport capacity that is not accompanied by changes on protein levels [ 14 , 16 ]. Therefore, as a next step we investigated whether chemical hypoxia for 24 h may regulate NBCe1 transport activity, regardless of regulation of NBCe1 protein abundance, as is the case in MES-like in dependent GBM cells.…”

Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na+ Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia

“…Additionally, a substrate-mediated increase in transport activity, fast and efficient regulation of NBCe1 transport may occur via post-translational modifications such as phosphorylation or by changes in transporter trafficking to the plasma membrane, without changes in transcript or protein expression [29]. In the context of the present work, it is noteworthy that brain ischemia causes regulation of mTOR signaling (reviewed by [87]), which is known to be able to phosphorylate several residues of NBCe1, thereby altering its transport activity [88,89].…”