Kidney Week

Abstract: TH-PO0050

The Role of Charged Residues in the Autostimulatory Domain of NBCe1-A

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
  October 22, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Jay, Gabriel, University at Buffalo, Buffalo, New York, United States

Gabriel Jay, MS

• Parker, Mark, University at Buffalo, Buffalo, New York, United States

Mark Parker, PhD

• Yeckley, Megan E., University at Buffalo, Buffalo, New York, United States

Megan E. Yeckley, MSc

Background

Mutations in the SLC4A4 gene that encodes the Na⁺/CO₃^2- cotransporter (NBCe1) result in phenotypic proximal renal tubular acidosis (pRTA). The N-terminal domain of the renal isoform NBCe1-A contains a unique 41-amino-acid autostimulatory domain (ASD) to the removal of which decreases single oocyte conductance by 64%. We hypothesize that charged residues in the ASD contribute to this phenomenon. In the present study, we determine the importance of charged side chains by systematically replacing them with pseudo-randomized uncharged amino acids.

Methods

We injected cRNA encoding either full-length wild-type (WT) or ASD-mutant human NBCe1-A or into Xenopus laevis oocytes. All constructs were engineered to include an HA-tag in their third extracellular loop. The three constructs being examined are the “No E” construct (E4S, E7A, and E15A), the “No RK” construct (K9S, R16S, R18A, and R20S), and the ASD-less (Δ41) mutant. Electrogenic Na⁺/CO₃^2- activity was assessed as CO₃-dependent conductance (G_CO3) using the two-electrode voltage clamp technique. Plasma membrane abundance relative to WT-NBC (T_NBC) was assessed via single-cell chemiluminescence detection of anti-HA immunoreactivity in fixed, intact cells. Thus G_CO3/T_NBC is our index of per-molecule activity relative to wild-type NBC.

Results

Analysis was performed through one-way ANOVA with Tukey’s correction for variance. This showed that the No E construct had significantly decreased (60%) per-molecule conductance compared to WT, which is not significantly different from the Δ41 mutant (60% decrease compared to WT). However, removing lysine and arginine residues resulted in a significant increase (105%) in per-molecule conductivity compared to WT.

Conclusion

Charged residues in the ASD of NBCe1-A are important for regulating protein activity. Negatively charged residues are important for upregulation of ion transport, while positively charged residues inhibit transport.

Funding

• Other U.S. Government Support