K+/Cl- cotransporter 2 (KCC2) and Na+/HCO3- cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.

K+/Cl- cotransporter 2 (KCC2) is a major Cl- extruder in mature neurons and is responsible for the establishment of low intracellular [Cl-], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl-], Cl- extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction-a mechanism with putative pathophysiological relevance.

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

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-1α protein levels, was mediated by TGF-ß signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1α-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.

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

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.

STAT3-dependent regulation of the electrogenic Na+/ HCO3- cotransporter 1 (NBCe1) functional expression in cortical astrocytes.

The electrogenic Na+ /HCO3- 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 (pHo ) 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 pHo 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 pHo -induced increased NBCe1 protein expression was prevented in astrocytes from Stat3flox/flox ::GfapCre/+ mice. In vitro, basal and high pHo -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.

Functional expression of electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse cortical astrocytes is dependent on S255-257 and regulated by mTOR.

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.