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Abstract: TH-PO1023

Novel Point Mutation in NBCe1/SLC4A4 Displays Dominant-Negative Inhibition of Wild-Type NBCe1A Activity in Mammalian Cells and Xenopus Oocytes

Session Information

  • Acid Base: Basic
    November 02, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 701 Acid-Base: Basic

Authors

  • Rossano, Adam Joseph, Mayo Clinic College of Medicine, Rochester, Minnesota, United States
  • Holmes, Heather L., Mayo Clinic College of Medicine, Rochester, Minnesota, United States
  • Romero, Michael F., Mayo Clinic College of Medicine, Rochester, Minnesota, United States
Background

NBCe1 (electrogenic Na+/HCO3- cotransporter 1; SLC4A4) is present at the proximal tubule basolateral membrane where it controls systemic pH by reclaiming HCO3-. Human recessive NBCe1 mutations cause proximal renal tubular acidosis, cataracts, and glaucoma. A recently discovered novel point mutation (NBCe1-Mut) produces similar clinical findings, but its biophysical characterization is lacking.

Methods

NBCe1A activity was assessed by voltage clamp in Xenopus oocytes. Membrane trafficking was followed using HA-tagged constructs. NBCe1A activity was also assayed in human trabecular meshwork cells (NTM-5) and retinal pigment epithelia (RPE) by co-transfecting GFP-tagged NBCe1 constructs with pHire (RFP-based pH indicator) to monitor intracellular pH.

Results

In oocytes, CO2/HCO3- (CB) addition elicited strong inward currents with NBCe1A-WT (WT). Currents in E91R (low-function) and Mut injected oocytes were indistinguishable from water controls. WT/E91R coinjections produced currents similar to WT, but co-injection of WT/Mut reduced maximum current to ~35% of WT-alone. HA tracking revealed that Mut trafficking to the plasma membrane was impaired compared to WT/E91R, and co-injection of WT/Mut constructs decreased successful trafficking to ~50% of WT-alone. Exposure of human cells expressing pHire to CB solutions produced a characteristic fall in pH followed by a gradual Na+-sensitive pH increase (HCO3- entry) via endogenous NBCe1. Cells transfected with WT displayed more rapid HCO3- entry than neighboring untransfected cells as well as dramatic acidification upon Na+ removal. pH transients in E91R transfected cells were indistinguishable from those in untransfected neighbors while expression of Mut slowed HCO3- entry and diminished the rate of acidification upon Na+ removal in both cell types studied.

Conclusion

NBCe1A-Mut displays poor transport and trafficking to the plasma membrane. Surprisingly, Mut can impair WT function and localization when co-expressed in amphibian and human cells. These results suggest a unique dominant-negative phenotype which requires further mechanistic analysis.

Funding

  • NIDDK Support