The electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), is the major bicarbonate transporter expressed in astrocytes. It is highly sensitive for bicarbonate and the main regulator of intracellular, extracellular, and synaptic pH, thereby modulating neuronal excitability. However, despite these essential functions, the molecular mechanisms underlying NBCe1‐mediated astrocytic response to extracellular pH changes are mostly unknown. Using primary mouse cortical astrocyte cultures, we investigated the effect of long‐term extracellular metabolic alkalosis 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 downregulation of NBCe1 activity following metabolic alkalosis without influencing protein abundance or surface expression of NBCe1. During alkalosis, the rate of intracellular H+ changes upon challenging NBCe1 was decreased in wild‐type astrocytes, but not in cortical astrocytes from NBCe1‐deficient mice. Alkalosis‐induced decrease of NBCe1 activity was rescued after activation of mTOR signaling. Moreover, mass spectrometry revealed constitutively phosphorylated S255‐257 and mutational analysis uncovered these residues being crucial for NBCe1 transport activity. Our results demonstrate a novel mTOR‐regulated mechanism by which NBCe1 functional expression is regulated. Such mechanism likely applies not only for NBCe1 in astrocytes, but in epithelial cells as well.

The electrogenic Na + /HCO 3 − cotransporter (NBCe1) in astrocytes is crucial in regulation of acid-base homeostasis in the brain. Since many pathophysiological conditions in the brain have been associated with pH shifts we exposed primary mouse cortical and hippocampal astrocytes to prolonged low or high extracellular pH (pH o) at constant extracellular bicarbonate concentration and investigated activation of astrocytes and regulation of NBCe1 by immunoblotting, biotinylation of surface proteins, and intracellular H + recordings. High pH o at constant extracellular bicarbonate caused upregulation of NBCe1 protein, surface expression and activity via upregulation of the astrocytic activation markers signal transducer and activator of transcription 3 (STAT3) signaling and glial fibrillary acidic protein expression. High pH o-induced increased NBCe1 protein expression was prevented in astrocytes from Stat3 flox/flox ::Gfap Cre/+ mice. In vitro, basal and high pH o-induced increased NBCe1 functional expression was impaired following inhibition of STAT3 phosphorylation. These results provide a novel regulation mode of NBCe1 protein and activity, highlight the importance of astrocyte reactivity on regulation of NBCe1 and implicate roles for NBCe1 in altering/modulating extracellular pH during development as well as of the microenvironment at sites of brain injuries and other pathophysiological conditions.

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.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells. Intracellular pH in tumour cells is alkaline, whereas extracellular pH is acidic. Consequently, the function and/or expression of pH regulating transporters might be altered. Here, we investigated protein expression and regulation of the electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) in mesenchymal (MES)-like hypoxia-dependent and -independent cells, as well as in astrocyte-like glioblastoma cells following chemical hypoxia, acidosis and elucidated putative underlying molecular pathways. Immunoblotting, immunocytochemistry, and intracellular pH recording with the H+-sensitive dye 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein were applied. The results show NBCe1 protein abundance and active NBCe1 transport. Hypoxia upregulated NBCe1 protein and activity in MES-like hypoxia-dependent GBM cells. This effect was positively correlated with HIF-1a protein levels, was mediated by TGF-b signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1a-independent manner. These results demonstrate a cell-specific adaptation of NBCe1 expression and activity to the microenvironment challenge of hypoxia and acidosis that depends on their transcriptional signature in GBM.

The electrogenic sodium bicarbonate co‐transporter 1 (NBCe1, Slc4a4) is a key pH‐regulating membrane protein and is widely expressed in kidney, pancreas, cornea, heart and brain. Phosphorylation in several residues can affect the surface expression and the activity of the transporter. NBCe1 has been found to be phosphorylated in several residues among them in Ser65 where SPAK inhibits its activity and in Thr49required for regulation of NBCe1 by IRBIT and SPAK. Moreover, IRBIT regulates the activity of NBCe1 by controlling the phosphorylation status of Ser232, Ser233 and Ser235. The aim of this study is to identify novel phosphorylation sites and examine their putative contribution in the transport activity of NBCe1. Phosphoproteomic analysis in mouse cortical astrocytes revealed constitutively phosphorylated NBCe1 at Ser245 and Ser255‐257. Mutational analysis in Hela cells and intracellular H+ recordings using pH‐sensitive fluorescent dye (BCECF) showed that the activity of NBCe1 is regulated by the phosphorylation of these residues, phospho‐Ser245 significantly reduced the activity of NBCe1 whereas phospho‐Ser255‐257led to increased NBCe1 activity. Additionally, in the presence of the mTOR inhibitor rapamycin or the mTOR activator 3BDO our data demonstrated that mTOR signaling pathway regulates the phosphorylation of the Ser245 and Ser255‐257. Our findings reveal phosphorylation at Ser245 and Ser255‐257as novel regulators of NBCe1 transport activity. We suggest that mTOR pathway is capable to fine tune NBCe1 activity, an event with putative implications during pathophysiological conditions.