Abstract: SA-PO1057

Functional Human Epithelial Na+ Channel Variants in the Extracellular Beta-Ball Domain

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Sheng, Shaohu, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Chen, Jingxin, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Kleyman, Thomas R., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

Epithelial Na+ channels (ENaC) have a key role in the regulation of extracellular fluid volume, extracellular K+ concentration and blood pressure. Recent human genome sequencing has revealed a large number of ENaC variants. However, the functional consequences of the vast majority of human ENaC variants are unknown. In this study, we investigated several non-synonymous ENaC variants located at a beta strand within a core beta-ball structure of the extracellular domain for their functional roles.

Methods

Point mutations corresponding to the selected variants were introduced into human alpha ENaC cDNA by site-directed mutagenesis. Wild type (WT) and mutant alpha subunits, together with WT beta and gamma subunits of human ENaC were expressed in Xenopus oocytes by cRNA injections. Channel activities were examined by two-electrode voltage clamp. Channel densities in plasma membranes were examined by a luminescence assay using a FLAG epitope tag inserted into the extracellular domain of beta subunit. Na+ self-inhibition was determined by measuring the decrease in current from the peak to the steady state elicited by a rapid increase in extracellular Na+ concentration from 1 to 110 mM at -100 mV.

Results

We examined three ENaC variants located at the beta strand 7, one of the five beta-ball strands at the extracellular domain core. Oocytes expressing the R350W ENaCs showed two-fold greater amiloride-sensitive currents than cells expressing WT channels (p < 0.001). The variation did not significantly alter channel surface expression. The mutant channels showed a diminished Na+ self-inhibition, which correlates to an increased open probability. The V351A mutant had a reduced current (55% of WT, p < 0.001), whereas G355R showed an increased current (1.6-fold of WT, p < 0.01).

Conclusion

R350W and G355R are gain-of-function ENaC variants, and V351A is a loss-of-function variant. R350W is an ENaC gating modifier via suppressing Na+ self-inhibition. Our results suggest that the core beta strand containing these variants and residing at a subunit interface has an important role in the regulation of ENaC gating.

Funding

  • NIDDK Support