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Int J Mol Sci
2021 Nov 26;2223:. doi: 10.3390/ijms222312817.
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Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3- Cotransporter NBCe1.
Alsufayan TA
,
Myers EJ
,
Quade BN
,
Brady CT
,
Marshall A
,
Haque N
,
Duffey ME
,
Parker MD
.
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In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+-2HCO3- stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3- influx. However, NBCe1 in the kidney mediates HCO3- efflux (HCO3- reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3- efflux) than in murine colons (in which NBCe1 mediates HCO3- influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.
Figure 1. Predicted phosphorylation sites in the cytoplasmic C-terminus of NBCe1. (A) The mode of action of NBCe1-A in the basolateral membranes of renal proximal tubule epithelia. HCO3− is generated in the cytosol by ammoniagenesis and the action of carbonic anhydrase II; (B) The mode of action of NBCe1-B in the basolateral membranes of colonic crypt epithelia. The process of HCO3− secretion into the lumen is completed via numerous apical anion transporters and channels [13]; (C) Graphic representation of NBCe1-A showing the cytoplasmic amino (Nt) and carboxy (Ct) terminal domains in relation to the transmembrane domain (TMD). Ct is not shown larger than scale in order to present the primary sequence. Highlighted residues were predicted to be phosphorylated (probability > 0.5) by the NetPhos 3.1 server (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 24 February 2021). Black highlighted residues 982 and 985 were demonstrated to be phosphorylated by a phosphoproteomic study of rat kidney cortices [14]. ATM = ATM kinase. PKA, PKB, PKC = protein kinases A, B, and C. cdc2 = cyclin-dependent kinase 1. cdk5 = cyclin-dependent kinase 5. CKII = casein kinase II. p38MAPK = p38 mitogen-activated kinases. RSK = ribosomal S6 kinases; (D) The sequence of mutant clones studied in the present work.
Figure 2. Validation of the pSer982 antibody. Western blot showing anti-pS982 immunoreactivity in murine kidney protein lysates (K = kidney lysate containing lane; — = blank lane). For the middle and right blots, the antibody was preabsorbed with peptides representing the non-phosphorylated version of the epitope (Pep1) with and without a peptide representing the phosphorylated epitope (Pep2).
Figure 3. Probing the pSer982 status of kidney and colonNBCe1. (A) Western blot showing anti-NBCe1 immunoreactivity and anti-pSer982 immunoreactivity in protein lysates from murine kidney (2 µg/lane) or colon (10 µg/lane); (B) Quantification of the ratio of pSer982/NBCe1 in colon versus kidney. Open circles represented null data points due to undetectable pSer982 immunoreactivity in colon and were excluded from the statistical analysis shown.
Figure 4. I–V relationships for oocyte expressing (de)phosphomimetic NBCe1-A clones. Each panel shows data gathered from a single representative oocyte injected with either (A) H2O, (B) cRNA-encoded wild-type NBCe1-A, or (C–F) cRNA encoded with one of five (de)phosphomimetic mutants. Open circles are I–V data gathered from oocytes during perfusion with our CO2/HCO3−-free solution, gray squares are I–V data gathered during perfusion with our CO2/HCO3−-containing solution, and black squares are I–V data gathered during perfusion with our CO2/HCO3−-containing solution plus 200 µM DIDS.
Figure 5. The conductance and stoichiometry of (de)phosphomimetic NBCe1-A clones in Xenopus oocytes. (A) The membrane conductance of a larger number of oocytes calculated from data, such as those shown in Figure 4. Letters above bars indicate statistically indistinguishable groups (i.e., groups with different letters were significantly different from each other); (B) The reversal potentials of wild-type NBCe1-A-EGFP and the four (de)phosphomimetic mutants plotted against the reversal potentials that correspond to predicted transport stoichiometries of 1Na+–3HCO3− (q = 3) and 1Na+–2HCO3− (q = 2).
Figure 6. The relative membrane abundance of (de)phosphomimetic NBCe1-A clones in Xenopus oocytes. (A) Western blot showing EGFP immunoreactivity in biotinylated protein fractions (i.e., cell-surface-expressed) NBCe1-A-EGFP from Xenopus oocytes; (B) Quantification of the relative plasma-membrane expression of (de)phosphomimetic mutants of NBCe1-A, as compared to wild-type. Letters above bars indicate statistically indistinguishable groups (i.e., groups with different letter were significantly different from each other).
Figure 7. I–V relationships for oocyte expressing (de)phosphomimetic NBCe1-B clones. Each panel shows data gathered from a single representative oocyte injected with either (A) H2O, (B) cRNA-encoded wild-type NBCe1-B-EGFP, or (C–F) one of five cRNA-encoded (de)phosphomimetic mutants. Open circles are I–V data gathered from oocytes during perfusion with our CO2/HCO3−-free solution, gray squares are I–V data gathered during perfusion with our CO2/HCO3−-containing solution, and black squares are I–V data gathered during perfusion with our CO2/HCO3−-containing solution plus 200 µM DIDS.
Figure 8. The conductance and stoichiometry of (de)phoshomimetic NBCe1-B clones in Xenopus oocytes. (A) The membrane conductance of a larger number of oocytes calculated from data, such as those show in Figure 7. Letters above bars indicate statistically indistinguishable groups (i.e., groups with different letter were significantly different from each other); (B) The reversal potentials of wild-type NBCe1-B and the four (de)phosphomimetic mutants plotted against the reversal potentials that correspond to predicted transport stoichiometries of 1Na+–3HCO3− (q = 3) and 1Na+–2HCO3− (q = 2).
Figure 9. The influence of 24 h metabolic acidosis on the pSer982 status of renal NBCe1 protein. (A) Western blots showing anti-NBCe1 and anti-pSer982 immunoreactivity (dimer (d), and monomer (m)) in mice that had ad libitum access to drinking water containing 0.5% sucrose (control) or 0.5% sucrose plus 0.28 M NH4Cl (metabolic acidosis); (B) Quantification of the ratio of pSer982/NBCe1 in control versus metabolic acidotic mice. Equal protein loading and transfer among blots was confirmed using MemCode reversible (total) protein stain (see Supplemental Figure S3).
Supplemental Figure S1
Western blot of mouse kidney protein probed with the anti-pSer982 antibody showing immunodepletion by pSer982 peptide (Pep2) as well as pS2r928/pSer985 peptide (Pep3)
Supplemental Figure S2 Western blot of mouse colon protein probed with the anti-SLC4A4 antibody showing lack of NBCe1 immunoreactivity in the colons of NBCe1b/c-null mice
Supplemental Figure S3 Total-protein stained PVDF membranes of western blots from control and acid-challenged groups
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