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

Abstract

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.

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

“…To determine the onset of high pH o ‐induced NBCe1 protein upregulation in mouse primary cortical astrocytes, cells were exposed to high pH o from 30 min to 12 hr, and subsequently NBCe1 protein abundance was determined by immunoblot analysis (Figure 2a). Using an antibody raised against NBCe1, specific immunoreactive bands at ~110 and ~130 kDa, were detected in controls, as previously reported (Khakipoor et al, 2019). Following exposure of astrocytes to high pH o for 6 hr, NBCe1 protein expression was significantly upregulated (Figure 2b; 1.15 ± 0.00 fold, * p < .05, using the two‐tailed unpaired Student's t ‐test, n = 3), compared to the controls (i.e., exposure to pH o 7.4), whereas after exposure for 4 hr, although increased (1.18 ± 0.10 fold) as well, the difference did not reach statistical significance.…”

“…To determine the onset of high pH o -induced NBCe1 protein upregulation in mouse primary cortical astrocytes, cells were exposed to high pH o from 30 min to 12 hr, and subsequently NBCe1 protein abundance was determined by immunoblot analysis (Figure 2a). Using an antibody raised against NBCe1, specific immunoreactive bands at~110 and 130 kDa, were detected in controls, as previously reported (Khakipoor et al, 2019). Following exposure of astrocytes to high pH o for 6 hr, NBCe1 protein expression was significantly upregulated (Figure 2b;…”

“…For primary culture of mixed glia, mouse pups aged P2/P3 were used, according to McCarthy and de Vellis (1980), as previously described (Khakipoor et al, 2019; Schrödl‐Häußel et al, 2015). Hippocampi and cortex were dissected and immediately collected in cold HBSS on ice.…”

“…Primary hippocampal and cortical astrocytes were harvested and homogenized, and protein concentration was determined according to Bradford (1976). Electrophoresis and blotting procedures were performed as described (Khakipoor et al, 2019). Primary antibodies were diluted as follows: NBCe1 1:1,000, Na + /K + ‐ATPase 1:1,000, GFAP 1:1,000, Vinculin 1:2,000, STAT3 1:1,000, pSTAT3 1:1,000, and GAPDH 1:10,000.…”

“…Astrocytic NBCe1 activity is shown to be regulated by several stimuli, among them acute extracellular pH changes, metabolic alkalosis, growth factors, and 4‐aminopyridine (4AP; Khakipoor et al, 2017; Salameh, Ruffin, & Boron, 2014; Schrödl‐Häußel, Theparambil, Deitmer, & Roussa, 2015; Theparambil et al, 2015, 2017). At the molecular level, NBCe1 associated cellular responses within or outside the CNS can be mediated either through direct phosphorylation (J. H. Hong et al, 2013; Khakipoor et al, 2019; Vachel et al, 2018; Yang et al, 2011) or/and by activation of several signaling pathways, such as mTOR, JNK, and Src/ERK signaling (Khakipoor et al, 2019; Namkoong et al, 2015; Schrödl‐Häußel et al, 2015).…”