Functional assessment of PIP 2 regulation of ENaC using an optogenetic dimerization assay

Abstract

The epithelial Na+ channel (ENaC) plays a key role in Na+ transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na+ reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP2 binding sites, by controlled depletion of PIP2. We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. CoroNa Green, a fluorescent Na+ indicator, was used to monitor ENaC activity by tracking the relative intracellular Na+ levels. Upon optogenetic-controlled depletion of PIP2, Na+ levels decreased in cells expressing wt ENaC. Mutations to the PIP2 sites of ENaC were expected to have no change in Na+ levels upon PIP2 depletion due to the disruption of PIP2 binding. As a control, mutations to non-PIP2 binding sites were included, and were expected to have decreased Na+ levels similar to wt ENaC. Interestingly, mutation of each independent PIP2 site resulted in only a small decrease of intracellular Na+, compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP2 binding site, resulted in a significant increase of Na+ upon PIP2 depletion. These data suggest that the residues surrounding the PIP2 binding sites play a significant role in the affinity of PIP2 for ENaC. The role of these other domains in PIP2 binding is still under investigation.