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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, 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 N termini of β- (K_d ∼5.2 μm) and γ-ENaC (K_d ∼13 μm). A weaker interaction site within the carboxyl terminus of γ-ENaC (K_d ∼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.

SUBMITTER: Archer CR

PROVIDER: S-EPMC7278353 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.

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Phosphatidylinositol 4,5-bisphosphate directly interacts with the β and γ subunits of the sodium channel ENaC.

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