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An international collaboration of the Potter Group with Solomon Snyder’s Group in the USA and with Chinese and German colleagues has uncovered the myo-inositol polyphosphate pyrophosphate 5-InsP7 as a regulator of the Na⁺/K⁺- ATPase-α1

Inositol hexakisphosphate kinase 1 associates with PI 3-kinase PI3K p85α and generates a local pool of 5-InsP7, which binds the RhoGAP domain of PI3K p85α. 5-InsP7 Binding disinhibits the PI3K p85α interaction with Na⁺/K⁺-ATPase-α1, recruiting AP2 that mediates clathrin-mediated endocytosis leading to downstream degradation of Na⁺/K⁺-ATPase-α1. The structure of 5-InsP7 is illustrated.
Model for 5-InsP7 regulation of Na⁺/K⁺-ATPase-α1 endocytosis and downstream degradation. Inositol hexakisphosphate kinase 1 associates with PI 3-kinase PI3K p85α and generates a local pool of 5-InsP7, which binds the RhoGAP domain of PI3K p85α. 5-InsP7 Binding disinhibits the PI3K p85α interaction with Na⁺/K⁺-ATPase-α1, recruiting AP2 that mediates clathrin-mediated endocytosis leading to downstream degradation of Na⁺/K⁺-ATPase-α1. The structure of 5-InsP7 is illustrated.

The sodium/potassium-transporting ATPase (Na⁺/K⁺-ATPase) is essential for eukaryotes and one of the most abundant cell membrane proteins. Universally expressed in the plasma membrane it generates electrochemical gradients across the cell membrane crucial for cell volume maintenance, signal transduction and the secondary transport of various nutrients. Endogenous negative regulators have for decades been postulated to play an important role in regulating the activity and stability of Na⁺/K⁺-ATPase, but their characterization has been elusive.

Our multi-disciplinary international collaboration has discovered that diphosphoinositol 1,2,3,4,6-pentakisphosphate, a higher myo-inositol polyphosphate pyrophosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream  degradation of Na⁺/K⁺-ATPase-α1. Deletion of IP6K1 enriches Na⁺/K⁺-ATPase-α1 in plasma membranes of multiple tissues and cell types.

Importantly, employing a suite of synthetic chemical biology tools, it was discovered that 5-InsP7 binds the RhoGAP domain of PI3K p85α to disinhibit its interaction with Na⁺/K⁺-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers clathrin-mediated endocytosis of Na⁺/K⁺-ATPase-α1.

In this structure-activity study inositol hexakisphosphate, lacking the 5-position pyrophosphate did not increase the binding of PI3K p85α with Na⁺/K⁺-ATPase-α1, demonstrating its requirement.

To explore how the pyrophosphate moiety affects binding, the synthetic regioisomers 1-diphosphoinositol pentakisphosphate (1-InsP7), 3-diphosphoinositol pentakisphosphate (3-InsP7) and 5-InsP7 (with the pyrophosphate motif at either the 1-, 3- or 5-position of the inositol ring, respectively) were evaluated. 1-InsP7 did not affect binding, but 3-InsP7 and 5-InsP7 increased it, suggesting positioning to be important.Diphosphoinositol tetrakisphosphate with a pyrophosphate at the 5-position but lacking a phosphate at the 2-position did not enhance binding, demonstrating a requirement for this phosphate group in addition to the pyrophosphate moiety.

Because 5-InsP7 can function in principle by either binding or pyrophosphorylating target proteins, specially-designed synthetic non-hydrolyzable 5-InsP7 analogues closely mimicking its physicochemical and biochemical properties, but unable to transfer their β-phosphoryl groups were utilized. Binding was enhanced indicating that protein pyrophosphorylation is not involved. Our study identifies 5-InsP7 as an endogenous negative regulator of Na⁺/K⁺-ATPase-α1.

For the full paper see:

https://advances.sciencemag.org/content/advances/6/44/eabb8542.full.pdf