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Abstract: PO1076

A Potential Novel Variant of Slc4a4 Can Regulate the Functional Activity of the Electrogenic Na/HCO3 Cotransporter NBCe1-B

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Lee, Seong-Ki, Case Western Reserve University School of Medicine Department of Physiology and Biophysics, Cleveland, Ohio, United States
  • Michenkova, Marie, Case Western Reserve University School of Medicine Department of Physiology and Biophysics, Cleveland, Ohio, United States
  • Romero, Michael F., Mayo Clinic Department of Physiology and Biomedical Engineering, Rochester, Minnesota, United States
  • Occhipinti, Rossana, Case Western Reserve University School of Medicine Department of Physiology and Biophysics, Cleveland, Ohio, United States
  • Boron, Walter F., Case Western Reserve University School of Medicine Department of Physiology and Biophysics, Cleveland, Ohio, United States
Background

The electrogenic Na/HCO3 cotransporter (NBCe1) regulates intracellular pH in many tissues and elicits vectorial HCO3 flow across many epithelia. Five variants of NBCe1 have been identified: -A, mainly in kidneys; -B, ubiquitous; -C, in brain; -D/E, in mouse reproductive organs. Because the A/D and B/C/E variants are transcribed from two distinct promoters, they have different NH2-termini (Nt). The Romero Lab developed an isoform-specific knockout (KO) mouse of NBCe1-A/D by causing a frameshift mutation in the A/D variants' unique Nt region (Chen et al, JASN 25:71A, 2014). Fang et al found that NBCe1-B (e1B) is expressed in kidneys of both WT and KO mice, and that e1B expression increases with metabolic acidosis in KOs (AJP Renal, 2018). They reached this conclusion by RT-PCR-amplification of B-variant–specific bands from KO kidneys. However, these amplifications generated several unidentified bands. Intrigued by these additional bands, we repeated the RT-PCR in an attempt to identify them.

Methods

Using TA cloning, we determined the sequences of two of the unidentified bands: (1) partial Slc4a4 product missing exon 4, which would lead to a frameshift, and (2) partial Slc4a4 product missing exons 4 & 5, but remaining in-frame. Because Fang et al confirmed lack of C/D/E variants, we introduced mutations (1) and (2) in e1B and studied functional activity by two-electrode voltage-clamping in Xenopus oocytes. We also determined protein abundance/interaction by surface protein biotinylation. Inasmuch as e1B has low activity, we co-expressed WT e1B and/or mutant e1B with super-IRBIT (which lacks PP1 binding site).

Results

We found that neither mutant, alone, has activity even though Δexon 4/5-e1B interacts with super-IRBIT. However, Δexon 4/5-e1B has a dominant-negative (DN) effect on WT e1B. To test if these mutants are specific for KO mice, we inspected other WT tissues.

Conclusion

Contrary to our hypothesis, we found that e1B mutants (1) and (2) exist at least in kidneys, brain, and pancreas, leading us to conclude that cells could in principle regulate NBCe1-B activity by adjusting the amount of the novel DN variant Δexon 4/5-e1B.

Funding

  • NIDDK Support