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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

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Published on: August 8, 2016

Engineering an enhanced voltage-sensing phosphatase.

Akira Kawanabe¹, Natsuki Mizutani¹, Onur K Polat²

¹Laboratory of Integrative Physiology, Department of Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

The Journal of General Physiology

|March 14, 2020

Summary

Voltage-sensing phosphatases (VSPs) are tools to study phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in cells. An enhanced VSP (eVSP) was developed for more effective PI(4,5)P2 depletion, improving ion channel research.

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Area of Science:

Biochemistry
Molecular Biology
Cell Biology

Background:

Voltage-sensing phosphatases (VSPs) possess a voltage sensor and a phosphoinositide-modulating enzymatic domain.
VSP activity is controlled by membrane potential, enabling manipulation of cellular phosphatidylinositol (PI) levels.
The PI(4,5)P2 lipid is crucial for the function of various ion channels and transporters.

Purpose of the Study:

To develop an enhanced voltage-sensing phosphatase (VSP) for more efficient depletion of PI(4,5)P2 in mammalian cells.
To create an improved molecular tool for investigating the role of PI(4,5)P2 in cellular processes, particularly ion channel activity.

Main Methods:

Engineering of the Danio rerio VSP (Dr-VSP) by introducing an aromatic residue (L223F) to enhance phosphatase activity.
Fusion of modified Dr-VSP with the N-terminal region of Ciona intestinalis VSP for improved plasma membrane targeting, creating eVSP.
Utilizing eVSP to induce voltage-evoked PI(4,5)P2 depletion and assessing its impact on Kir2.1, KCNQ2/3, and TRPC6 channel activity.

Main Results:

The engineered eVSP exhibited enhanced voltage-dependent phosphatase activity compared to the wild-type Dr-VSP.
eVSP induced more pronounced and rapid changes in cellular PI(4,5)P2 levels upon voltage stimulation.
Functional readouts from Kir2.1, KCNQ2/3, and TRPC6 channels demonstrated significant alterations in their activity due to eVSP-induced PI(4,5)P2 depletion.

Conclusions:

The developed eVSP is a superior molecular tool for manipulating PI(4,5)P2 levels in living cells.
eVSP facilitates the study of PI(4,5)P2's role in regulating ion channel and transporter function.
This enhanced VSP offers greater precision and efficacy for investigating lipid signaling pathways.