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 (PIP
2
) 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 PIP
2
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 PIP
2
binding sites, by controlled depletion of PIP
2
. We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP
2
levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na
+
indicator, was used to monitor ENaC activity by tracking the relative intracellular Na
+
levels. Upon optogenetic-controlled depletion of PIP
2
, Na
+
levels decreased in cells expressing wt ENaC. Mutations to the PIP
2
sites of ENaC were expected to have no change in Na
+
levels upon PIP
2
depletion due to the disruption of PIP
2
binding. As a control, mutations to non-PIP
2
binding sites were included, and were expected to have decreased Na
+
levels similar to wt ENaC. Interestingly, mutation of each independent PIP
2
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 PIP
2
binding site, resulted in a significant increase of Na
+
upon PIP
2
depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na
+
fluctuations. These data suggest that the residues surrounding the PIP
2
binding sites play a significant role in the affinity of PIP
2
for ENaC. The role of these other domains in PIP
2
binding is still under investigation.