Regulation of electrogenic Na+/HCO3- cotransporter 1 (NBCe1) in mouse cortical astrocytes and its dependence on m-TOR mediated phosphorylation of Ser245.
- Organism: Mus musculus
- Instrument: Q Exactive HF-X
glial cells, acid-base, pH, signaling, acidosis
Lab head: Christina Ludwig
Submitter: Christina Ludwig
Astrocytes are pivotal responders to alterations of extracellular pH, primarily by regulation of their “master” acid-base transporter, the membrane-bound electrogenic Na+/bicarbonate cotransporter 1 (NBCe1). Here, we describe an 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 (BCECF), and phosphoproteomic analysis. The results showed significant increase of NBCe-1-mediated recovery of intracellular pH from acidification in wild-type astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Acidosis-induced upregulated NBCe1 activity was prevented following inhibition of mTOR signaling by rapamycin. In contrast, during acidosis or following exposure of astrocytes to rapamycin, surface protein abundance of NBCe1 remained comparable. Furthermore, NBCe1 activity was dependent on phosphorylation state of Ser245, a residue conserved in all NBCe1 variants. Mutational analysis suggested that 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.
Primary cortical astrocytes were subjected to control or extracellular acidosis for 30 min, 3 h or 6 h in the presence or absence of 1µM rapamycin and then kept on ice. Isolation of cell surface proteins was performed using the Pierce® cell surface protein isolation kit following the manufacturer’s instructions. Probes were then processed for immunoblotting with antibodies against NBCe1, Na+/K+-ATPase, and GAPDH, or for quantitative phosphoproteomic analysis.
After surface-proteome enrichment all samples were dissolved in 1x NuPAGE LDS sample buffer (ThermoFisher Scientific). In-gel trypsin digestion was performed according to standard procedures. Briefly, the samples were run on a NuPAGETM 4-12% Bis-Tris protein gel (ThermoFisher Scientific) for 5 min. Subsequently, the still not size-separated single protein band per sample was cut, reduced (50 mM dithiothreitol), alkylated (55 mm chloroacetamide) and digested overnight with trypsin (Trypsin Gold, Promega). The generated peptides were dried in a vacuum concentrator and dissolved in 25 μL 2% (v/v) acetonitrile, 0.1% (v/v) formic acid in HPLC grade water.
LC-MS/MS measurements were performed on an Ultimate 3000 RSLCnano system coupled to a Q-Exactive HF-X mass spectrometer (ThermoFisher Scientific). For each analysis, 5 μL of peptides were delivered to a trap column (ReproSil-pur C18-AQ, 5 μm, Dr. Maisch, 20 mm × 75 μm, self-packed) at a flow rate of 5 μL/min in 100% solvent A (0.1% (v/v) formic acid in HPLC grade water). After 10 minutes of loading, peptides were transferred to an analytical column (ReproSil Gold C18-AQ, 3 μm, Dr. Maisch, 400 mm × 75 μm, self-packed) and separated using a 50 min gradient from 4% to 32% of solvent B (0.1% (v/v) formic acid, 5% (v/v) DMSO in acetonitrile) at 300 nL/min flow rate. nanoLC solvent A was 0.1% (v/v) formic acid, 5% (v/v) DMSO in HPLC grade water. The Q-Exactive HF-X mass spectrometer was operated in data dependent acquisition and positive ionization mode. MS1 spectra (360–1300 m/z) were recorded at a resolution of 60,000 using an automatic gain control (AGC) target value of 3e6 and maximum injection time (maxIT) of 45 ms. Up to 18 peptide precursors were selected for fragmentation. Only precursors with charge state 2 to 6 were selected and dynamic exclusion of 25 sec was enabled (mass tolerance +/-10 ppm). Peptide fragmentation was performed using higher energy collision induced dissociation (HCD) and a normalized collision energy (NCE) of 26%. The precursor isolation window width was set to 1.3 m/z. MS2 Resolution was 15.000 with an automatic gain control (AGC) target value of 1e5 and maximum injection time (maxIT) of 25 msec.
Peptide identification and quantification was performed using the software MaxQuant (version 126.96.36.199) with its built-in search engine Andromeda. MS2 spectra were searched against the mus musculus reference protein database from UniProt (UP000000589, 17038 reviewed protein entries, download 17.7.2020), supplemented with common contaminants (built-in option in MaxQuant). Trypsin/P was specified as proteolytic enzyme. Carbamidomethylated cysteine was set as fixed modification. Phosphorylation on serine, threonine and tyrosine was specified as variable modification, as well as oxidation of methionine and acetylation at the protein N-terminus. Precursor tolerance was set to 4.5 ppm, and fragment ion tolerance to 20 ppm. Results were adjusted to 1 % false discovery rate (FDR) on peptide spectrum match (PSM) level and protein level employing a target-decoy approach using reversed protein sequences. The minimal peptide length was defined as 7 amino acids, the “match-between-run” function was disabled. A targeted quantitative analysis of all identified NBCe1 peptides (phosphopeptides as well as naked peptides) was performed using the software Skyline. Extracted precursor ion chromatograms were integrated and the resulting NBCe1 phosphopeptide intensities were normalized to the total NBCe1 protein intensity (summed up intensities of all naked peptides) in each sample respectively. The corresponding Skyline document, as well as all proteomic raw data and MaxQuant search results have been deposited to Panorama Public.
Created on 3/4/21, 9:49 AM