Intracellular Ionic Strength Sensing Using NanoLuc.

Tausif Altamash,Kabir H Biswas,Wesam Ahmed,Saad Rasool

doi:10.3390/ijms22020677

Tausif Altamash, Kabir H Biswas + Show 2 more

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https://doi.org/10.3390/ijms22020677

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Abstract

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

Highlights

Ionic strength determined by the concentrations of different ionic species in a medium, both cations and anions, fundamentally influences electrostatic interactions in and among biomolecules and, regulates a plethora of cellular processes [1,2,3,4]
We have previously found that the bioluminescence of Renilla luciferase (Rluc) is affected by the ionic strength [7], and hypothesized that this ionic strength effect can be extrapolated to other luciferases and used for ionic strength sensing
We have attempted to develop an ionic strength sensing strategy using the NanoLuc protein. This was premised on the observation of increased bioluminescence that we had reported for the Rluc protein in cell lysates previously [7]

Summary

Introduction

Ionic strength determined by the concentrations of different ionic species in a medium, both cations and anions, fundamentally influences electrostatic interactions in and among biomolecules and, regulates a plethora of cellular processes [1,2,3,4]. Variations in intracellular ionic strength can occur due to changes in the osmolarity of the cellular microenvironment, which may result in either osmotic swelling or shrinkage of cells. This can subsequently lead to the activation of regulatory volume decrease or increase mechanisms, respectively, through switching of volume-sensitive ion channels [22]. Intracellular signaling events, such as those leading to changes in the metabolic activity of the cell, can affect intracellular ionic strength. Arachidonic acid metabolites can activate certain potassium channels and inhibit voltage-sensitive chloride channels [22]

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