1.
Optogenetic Control of PIP2 Interactions Shaping ENaC Activity.
Abstract:
The activity of the epithelial Na+ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of β- and γ-ENaC subunits (βN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na+ levels [Na+]i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na+]i by measuring the fluorescent Na+ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to βN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant βN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na+]i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the βN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.
2.
Phosphatidylinositol 4,5-bisphosphate directly interacts with the β and γ subunits of the sodium channel ENaC.
Abstract:
The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na+ channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, though, remains obscure. To date, possible interaction of PIP2 with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the amino termini of β- (Kd ~5.2 µM) and γ-ENaC (Kd ~13 µM). A weaker interaction site within the carboxy terminus of γ-ENaC (Kd ~800 µM) was also observed. These results support that PIP2 regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.
